the architecture of parallel β-helices and related folds


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Page 1: The architecture of parallel β-helices and related folds

Progress in Biophysics & Molecular Biology 77 (2001) 111–175


The architecture of parallel b-helices and related folds

John Jenkinsa,*, Richard Pickersgillb

a Institute of Food Research, Norwich Research Park, Colney Lane, Norwich NR4 7UA, UKbBiological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK


Three-dimensional structures have been determined of a large number of proteins characterized by arepetitive fold where each of the repeats (coils) supplies a strand to one or more parallel b-sheets. Some ofthese proteins form superfamilies of proteins, which have probably arisen by divergent evolution from acommon ancestor. The classical example is the family including four families of pectinases withoutobviously related primary sequences, the phage P22 tailspike endorhamnosidase, chrondroitinase B andpossibly pertactin from Bordetella pertusis. These show extensive stacking of similar residues to givealiphatic, aromatic and polar stacks such as the asparagine ladder. This suggests that coils can be added orremoved by duplication or deletion of the DNA corresponding to one or more coils and explains howhomologous proteins can have different numbers of coils.

This process can also account for the evolution of other families of proteins such as the b-rolls, theleucine-rich repeat proteins, the hexapeptide repeat family, two separate families of b-helical antifreezeproteins and the spiral folds. These families need not be related to each other but will share features such asrelative untwisted b-sheets, stacking of similar residues and turns between b-strands of approximately901often stabilized by hydrogen bonding along the direction of the parallel b-helix.

Repetitive folds present special problems in the comparison of structures but offer attractive targets forstructure prediction. The stacking of similar residues on a flat parallel b-sheet may account for theformation of amyloid with b-strands at right-angles to the fibril axis from many unrelated peptides. r 2001Elsevier Science Ltd. All rights reserved.


1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

1.1. Scope of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1121.2. Nomenclature, definitions and general features of parallel b-helices . . . . . . . . . . . . 116

1.2.1. Parallel b-helix and its b-sheets . . . . . . . . . . . . . . . . . . . . . . . . . . 116

*Corresponding author.

E-mail address: [email protected] (J. Jenkins).

0079-6107/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 7 9 - 6 1 0 7 ( 0 1 ) 0 0 0 1 3 - X

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1. Introduction

1.1. Scope of the review

In 1993 Yoder et al. (1993a) reported the structures of the first parallel b-helix and Baumannet al. (1993) that of the first b-roll, which were at first regarded as revolutionary in using parallel

1.2.2. Coils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161.2.3. Stacks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

1.2.4. Turns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221.2.5. Packing of b-sheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

2. Description of known structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262.1. Pectinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

2.1.1. The extra-cellular pectate lyase family . . . . . . . . . . . . . . . . . . . . . . . 126

2.1.2. Polygalacturonases and rhamnogalacturonase A . . . . . . . . . . . . . . . . . . 1302.1.3. Pectin methylesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1332.1.4. PelL from Erwinia chrysanthemi . . . . . . . . . . . . . . . . . . . . . . . . . . 134

2.1.5. Pectate lyase Pel-15 from Bacillus sp. strain KSM-P15 . . . . . . . . . . . . . . 1342.2. The P22 phage tailspike endorhamnosidase . . . . . . . . . . . . . . . . . . . . . . . . . 1352.3. Chrondroltinase B from flavobacterium hepinarum . . . . . . . . . . . . . . . . . . . . . 138

2.4. P69 pertactin from Bordetella pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . 1392.5. Glutamate synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1402.6. The antifreeze protein from Tenebrio molitor . . . . . . . . . . . . . . . . . . . . . . . 141

2.7. The leucine-rich repeat family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422.7.1. Ribonuclease inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1432.7.2. The GTPase-activating protein Ma1P from Schizosaccharomyces pombe . . . . . 1442.7.3. Human insulin-like growth factor receptor . . . . . . . . . . . . . . . . . . . . . 144

2.7.4. Human spliceosomal protein U2A0, Rab geranylgeranyltransferase and themRNA export factor TAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

2.7.5. Internalin B from Listeria monocytogenes . . . . . . . . . . . . . . . . . . . . . 146

2.8. Left-handed parallel b-helix structures . . . . . . . . . . . . . . . . . . . . . . . . . . . 1472.8.1. Left-handed parallel b-helix structures containing hexapeptide repeats . . . . . . 1472.8.2. The left-handed parallel b-helix antifreeze protein from spruce budworm . . . . . 150

2.9. Parallel b-rolls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1512.10. Spiral folds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

3. The prediction and design of parallel b-helix structures . . . . . . . . . . . . . . . . . . . . . 152

4. Are amyloid fibrils related to parallel b-helices? . . . . . . . . . . . . . . . . . . . . . . . . . 156

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1615.1. Evolutionary relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

5.2. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

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b-sheet alone to form complex structures by repeating individual coils. Subsequently the group ofproteins built using similar principles, for which Jurnak et al. (1994) used the term coiled folds andKobe and Deisenhofer (1995a, b) used solenoid proteins, has greatly extended. However, newdiscoveries do not remain shocking for long and structural biology has rapidly absorbed thelessons of these structures. Purely parallel b-structures are no longer described as unstable and theparallel b-helix has simply taken its place amongst the globular proteins with all b-folds. This isjustified because the parallel b-helix architecture is simply one of many ways to form a globularfolded protein, which are stabilised by the same types of interactions. Thus we can easily imaginea parallel b-helical enzyme and one with a different architecture converging to use the samecatalytic mechanism.

However, structures with the parallel b-helix architecture do possess some unusual commonfeatures and the ambiguity of finding a unique solution when aligning these structures doespose special problems when comparing them. The unusual simplicity of the architecture alsosuggests that understanding their evolution or predicting their occurrence may be unusuallyeasy. This is not unique and may also apply to folds such as the b-propeller fold (F .ull .op andJones, 1999).

Parallel b-helix domains appear to fold as a single co-operative unit as do the domains of otherglobular proteins. However, there is a sense in which parallel b-helices and other coiled folds areintermediate between most globular enzymes and structures made up of domains arranged asbeads on a string, where the folding unit is the individual repeating unit. In the parallel b-helicesthe unit of folding is the domain while the unit of evolution may have been the individual ‘‘coil’’(see Section 1.2.2 below).

Parallel b-helices also form a bridge between globular and fibrous proteins. The rather flat(untwisted) parallel b-sheets and the ‘‘stacks’’ of similar or alternating residues are special andprobably related features of these folds and we may expect to find similarities between the parallelb-helix architecture and other systems where untwisted b-sheets pack against each other. This hasled to analogies being made between parallel b-helices and some models of amyloid.

Kobe and Kajava (2000) have recently briefly reviewed the whole family of coiled folds orsolenoid proteins and identified 18 solenoid folds (see The structures containing only a-helices have been recently reviewed by Kobe et al.(1999) and we will focus on the structures with parallel b-sheets, which are listed in Table 1, andespecially the parallel b-helical proteins. Even this is a long list and is growing rapidly (forexample two different parallel b-helical antifreeze protein families have been published in 2000,glutamate synthase shows a parallel b-helical domain and the abstract by Rozwarski et al. (1999)appears to promise a new family). The main focus will be the architecture of these proteins ratherthan the details of their function. All these parallel b-sheet containing proteins have somecommon architectural features. These may also share a common mechanism of evolution (asdistinct from a common ancestor). They show the more general properties of solenoid proteinsidentified by Kobe and Kajava (2000). However, the focus on the parallel b-sheet containingproteins makes it hard to discuss Kobe and Kajava’s suggestion that evolution may relatethe leucine-rich repeat (LRR) proteins discussed below with the LRR variant family with a3/10-helix and an a-helix per coil (Peters et al., 1996). We will also include a brief description ofthe evidence for proposed amyloid structures, especially on the suggestions of similarity to parallelb-helices.

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Table 1Structures with repetitive folds containing b-sheetsa

Name and origin of the protein Short name PDB code Resolution in (A,



Right-handed parallel b-helix proteins

Pectate lyase PelC from Erwinia PelC 1AIR 2.20 [1]

chrysanthemi 1PLU 2.20 [2] Lu3+ complex

Pectate lyase from Bacillus subtilis Bspel 1BN8 1.80 [3]

2BSP 1.80 R279K

Pectate lyase PelE from Erwinia


PelE 2.20 [2,4]

Pectin lyase PnlA from Aspergillus niger PnlA 1IDJ 2.40 [5] pH 6.5

1IDK 1.93 [5] pH 8.5

Pectin lyase PnlB from Aspergillus niger PnlB 1QCX 1.70 [6]

Rhamnogalacturone A from Aspergillus RGase A 1RMG 2.00 [7]


Polygalacturonase PehA from Erwinia


PehA 1BHE 1.90 [8]

Polygalacturonase II from Aspergillus


PG II 1CZF 1.68 [9]

Pectin methylesterase from Erwinia


PemA 1QJV 2.40 [10]

Pectate lyase Pel-15 from Bacillus

sp. strain KSM-P15

Pel-15 1EE6 2.30

Salmonella P22 phage tailspike TSP 1TSP 2.00 [11,12]

endorhamnosidase 1TYU 1.80 [13] Complex

1TYV 1.80 [13] Complex

1TYW 1.80 [13] Complex

1CLW 2.0 [14] V331A

1QA1 2.0 [14] V331G

1QA2 2.0 [14] A334V

1QQ1 1.8 [15] E359G

1QRB 2.0 [15] T326F

1QRC 2.5 [15] W391A

Chrondroitinase B from Flavobacterium 1DBG 1.70 [16]

hepinarum 1DBO 1.70 [16] Complex

P69 pertactin from Bordetella pertussis Pertactin 1DAB 2.60 [17]

Glutamate synthase from Azospirillum


1EAO 3.0 [18]

Antifreeze protein from Tenebrio molitor TmAFP 1EZG 1.40 [19]

Leucine-rich repeat proteins

Porcine ribonuclease inhibitor RI 2BNH 2.30 [20]

1DFJ 2.50 [21] Ribonuclease


Human ribonuclease inhibitor HRI 1A4Y 2.00 [22] Angiogenin


Human insulin-like growth factor receptor IGF 1IGR 2.60 [23]

Human spliceosomal protein U2A0 U2A0 1A9N 2.4 [24] U2b00-U2A0U2

RNA complex

Rab geranylgeranyltransferase RabGGT 1DCE 2.0 [25]

Human mRNA export factor TAP TAP 1FO1 2.9 [26]

GTPase-activating protein Ma1P from

Schizosaccharomyces pombe

Ma1P or


1YRG 2.66 [27]

Internalin B from Listeria monocytogenes InlB 1DOB 1.86 [28]

J. Jenkins, R. Pickersgill / Progress in Biophysics & Molecular Biology 77 (2001) 111–175114

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Table 1 (continued)

Name and origin of the protein Short name PDB code Resolution in (A,



Left-handed parallel b-helix hexapeptide

repeat proteins


acyltransferase from Escherichia coli

LpxA 1LXA 2.60 [29]

Carbonic anhydrase from

Methanosarcina thermophila

Cam 1THJ 2.80 [30]


N-succinyltransf erase

DapD 1TDT 2.20 [31]

2TDT 2.00 [32] Complex

3TDT 2.00 [32] Complex

Xenobiotic acetyltransferase from

Pseudomonas aeruginosa

PaXAT 1XAT 3.20 [33]

2.25 [34]


mine 1-phosphate uridyltransferase

from E. coli (truncated)

2.30 [34] Complex

N-acetylglucosamine 1-phosphate

uridyltransferase from E. coli

1HV9 2.1 [35] Complex

N-acetylglucosamine 1-phosphate 1G95 2.33 [36]

uridyltransferase from 1G97 1.96 [36] Complex

Streptococcus pneumoniae 1HMO 2.3 [37]

1HM8 2.5 [37] Complex

1HM9 1.75 [37] Complex

Left-handed parallel b-helix pentapeptide repeat proteins

Antifreeze protein from spruce budworm sbwAFP 1EWW NMR [38]

b-roll proteinsAlkaline protease from Pseudomonas


1KAP 1.64 [39]

50 kDa metalloprotease from Serratia


1SAT 1.75 [40]

Spiral folds

4-Chlorobenzoyl coenzyme A dehalogenase 1NZY 1.80 [41]

Enoyl-coenzyme A hydratase 1DUB 2.50 [42]

2DUB 2.40 [43] Complex

ATP-dependent Clp protease from

Escherichia coli

ClpP 1TYF 2.20 [44]

aPublications describing new or refined structures are given as [1] above. These are 1. Yoder et al. (1993a); 2. Lietzkeet al. (1994); 3. Pickersgill et al. (1994); 4. Lietzke et al. (1996); 5. Mayans et al. (1997); 6. Vitali et al. (1998); 7. Petersenet al. (1997); 8. Pickersgill et al. (1998); 9. Van Santen et al. (1999); 10. Jenkins et al. (2001); 11. Steinbacher et al. (1994);

12. Steinbacher et al. (1997); 13. Steinbacher et al. (1996); 14. Baxa et al. (1999); 15. Schuler et al. (2000); 16. Huang et al.(1999); 17. Emsley et al. (1996); 18. Binda et al. (2000); 19. Liou et al. (2000); 20. Kobe and Deisenhofer (1993); 21.Kobe and Deisenhofer (1995b); 22. Papageorgiou et al. (1997); 23. Garrett et al. (1998); 24. Price et al. (1998); 25. Zhanget al., 2000; 26. Liker et al. 2000; 27. Hillig et al. (1999); 28. Marino et al. (1999); 29. Raetz and Roderick (1995); 30.

Kisker et al. (1996); 31. Beaman et al. (1997); 32. Beaman et al. (1998a); 33. Beaman et al. (1998b); 34. Brown et al.(1999); 35. Olsen and Roderick (2001); 36. Kostrewa et al. (2001); 37. Sulzenbacher et al. (2001); 38. Graether et al.(2000); 39. Baumann et al. (1993); 40. Baumann (1994); 41. Benning et al. (1996); 42. Engel et al. (1996); 43. Engel et al.

(1998); 44. Wang et al. (1997).

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1.2. Nomenclature, definitions and general features of parallel b-helices

1.2.1. Parallel b-helix and its b-sheetsThe term ‘‘parallel b-helix’’ was introduced by Yoder et al. (1993a) when reporting the structure

of the pectate Lyase PelC, the first protein structure displaying this fold. It is important to notethat the ‘‘parallel b-helix’’ is not related to the term ‘‘b-helix’’, used earlier to describe thestructure of gramicidin (Wallace and Ravikumar, 1988; Langs, 1988). The publication of the firstleft-handed structure of UDP-N-acetylglucosamine acyltransferase by Raetz and Roderick (1995)required that ‘‘parallel b-helix’’ be further extended to either ‘‘right-handed parallel b-helix’’ or‘‘left-handed parallel b-helix’’. The nomenclature generally used to describe the basic architectureof the parallel b-helical proteins was essentially fixed by Yoder et al. (1993b), who introduced thenomenclature PB1, PB2 and PB3 for the three parallel b-sheets of pectate lyase and T1, T2 and T3(turn) for the regions following the b-sheets. This was revised to include PB1a and implicitly T1aby Petersen et al. (1997) when the structure of rhamnogalacturonase A from Aspergillus aculeatusclearly showed a fourth b-sheet. Figs. 1 and 2 illustrate this convention. An alternativenomenclature was proposed for a fragment of the P22 phage tailspike protein by Steinbacher et al.(1994) in which the PB1 is equivalent to b-sheet C, PB2 equivalent to A and PB3 equivalent to B.Van Santen et al. (1999) in describing polygalacturonase II from Aspergillus niger use PB2a andPB2b as an alternative nomenclature corresponding to PB1a and PB2 used for RGase A andPehA.

1.2.2. CoilsAs ‘‘turn’’ was used by Yoder et al. (1993b) to describe the regions between the b-stands by

analogy with b-turns in hairpin loops, there is a need for an unambiguous term for a completeturn of the parallel b-helix and the term ‘‘coil’’ is used in this review. However, there remains thedifficult problem of whether to count the coils using topological arguments or to accept only coilswith the regular three-stranded parallel b-helical structure. This ambiguity results in greatconfusion on how many coils occur in known structures and in how to number them. A goodexample is provided by the family of hydrolases containing RGase A, PehA and PG II because thereasoning for the different choices between 10 and 13 coils can be explicitly described. RGase Amight be said to have 12 coils starting with the first PB2 (residues 20–22) and finishing with the


Fig. 1. Ribbon diagrams and views of the cross-section of single coils running clockwise for various types of repetitivefold. The atoms of the cross-section are coloured by type: black for carbon, blue for nitrogen, red for oxygen and yellow

for sulphur. (a) Part of the b-roll of the alkaline protease from Pseudomonas aeroginosa (1 kap) with the tandemrepeated sequence GGXGXDXLX giving rise to the b-roll architecture. The two b-sheets are coloured red and green.Calcium ions are shown as yellow balls. The single coil runs clockwise starting with the sequence Ile–Leu–Tyr. (b)Bacillus subtilis pectate lyase (1bn8) with the N-terminal end of the parallel b-helix to the left and the T3 region above.

The three b-sheets PB1 (Thr–Asp–Ala), PB2 (Ile–Thr–Met) and PB3 (Tyr–Tyr–His) are coloured yellow, green and red.An asparagine of the asparagine ladder can be seen at T2 (just before PB3) and a residue of the aromatic stack can beseen on PB3. (c) Porcine ribonuclease inhibitor with the b-sheets in red and the helices in green. In the ribbon diagram

the chain direction or direction of the superhelix is anti-clockwise. However, the single coil runs clockwise and showsthe leucines from the a-helix and b-sheet packing in the hydrophobic core. (d) The left-handed UDP-N-acetylglucosamine acyltransferase from Escherichia coli with the N-terminal end of the parallel b-helix to the right.

The coil shows the isoleucines that form its hydrophobic core.

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last PB1a (residues 353–356) as shown in Table 1 of Petersen et al. (1997), which lists 12 PB2stands. Petersen et al. suggest that there are 13 coils because the main chain forms a very tight coilwith residues 354 and 356 forming hydrogen bonds to the previous PB2 at residues 331–333 (i.e.the last PB1a acts as both a PB1a and a PB2), forms a disulphide to Cys 350 in the previous PB1

Fig. 2. Ribbon diagrams of some of the longer right-handed parallel b-helices. The three common b-sheets PB1, PB2

and PB3 are coloured yellow, green and red, while the extra sheet PB1a found in the polygalacturonase family iscoloured blue. The N-terminal end of the right-handed parallel b-helices are to the right. (a) The Salmonella P22 phagetailspike endorhamnosidase. (b) P69 pertactin from Bordetella pertussis showing the longest known parallel b-helix. (c)

The polygalacturonase PehA from Erwinia carotavora showing the PB1a sheet within the T1 region.

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from Cys 359 and then reverses direction to give a last PB1 strand (residues 369–375), so that thetopological winding number is 13. However, the strand of residues 369–375 is anti-parallel to theprevious PB1 making the nomenclature PB1 rather confusing, so that we might reject the residuesafter 356 as not being part of the parallel b-helix. We might also reject the first coil because itcontains a single b-strand (PB2) and an a-helix by arguing that this is not a parallel b-helix,leaving us with 11 coils. Finally, using the original definition of Yoder et al. (1993b) and requiringthat each coil starts with PB1 and ends with PB3 would leave 10 coils, which is how the verysimilar structure of PehA is described by Pickersgill et al. (1998). PehA forms the same final PB1ato PB2 hydrogen bonds but does not form the disulphide or the anti-parallel structure. PGII doesnot form the PB1a to PB2 hydrogen bonds as it forms a rather different structure with proline 357in approximately a–R conformation at the end of PB1a (or PB2a), terminates by forming thedisulphide without any anti-parallel extension and is also described as having 10 complete turns(van Santen et al., 1999). What is more significant than the number of coils used to describe thestructure is that these hydolases have two complete coils and a PB1 and PB1a more than thepectate lyases or the pectin methylesterases and three fewer coils than the P22 phage tailspikeendorhamnosidase. It is important to check the number of coils visually rather than to acceptstatements in the literature since an unexpected definition may have been used.

1.2.3. StacksYoder et al. (1993b) first discussed the ‘‘stacking’’ of similar residues at the equivalent positions

in neighbouring coils and this was extended by Raetz and Roderick (1995), who revived the term‘‘cupped stacks’’ for the most common stacking of valines and isoleucines. Petersen et al. (1997)introduced the useful distinction between ‘‘stacked’’ and ‘‘aligned’’ residues. ‘‘Stacked’’ residueshad to be similar residues with similar w angles while ‘‘aligned’’ described the same or chemicallysimilar residues packing with different w angles (especially w1). The distinction is between regularand irregular structures but ‘‘stacks’’ that mix valines and isoleucines with leucines or methionineswith similar w angles do occur. The stacks of valines and isoleucines packing on each other tend tobe more regular. There are at least three different types of stacks found in the parallel b-helixproteins as illustrated in Figs. 3 and 4. Aliphatic stacks such as the ‘‘cupped stacks’’ are the mostcommon and are found in every protein, aromatic stacks are formed by the offset face to facepacking of phenylalanine and tyrosine side chains (Hunter et al., 1991) and polar stacks includethe well-known asparagine ladder (Yoder et al., 1993b), several variations of which are shown inFigs. 4 and 5.

‘‘Aligned’’ residues are ubiquitous in b-sheet containing proteins and occur in both parallel andanti-parallel sheets. ‘‘Cupped stacks’’ of valines and isoleucines were discussed by Richardson(1981) long before the parallel b-helix was observed, who noted that the type of packing seen inthe ‘‘stacks’’ will not be stable in anti-parallel b-sheet because it would demand that the w1 anglesdiffer by 1801. However, such stacking is rather rare except in the repetitive folds. In a highlytwisted parallel b-sheet, stacking will be restricted to a single ridge if it occurs, which may explainits rarity in TIM barrels. An example of a single ridge is seen in glyceraldehyde-3-phosphatedehydrogenase from Sulfolobus solfataricus, PDB code 1B7G (Isupov et al., 1999) where residuesIle 50, Val 31, Val 6, Val 82, Ile 105, Ile 134 and Ile 117 form a ridge and Val 6 to Ile 134 clearlyform a cupped stack. By contrast, the relationship between stacking and the repetitive folds is oneof the most basic features of their architecture so that no parallel b-helix has been observed

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without stacking. However, the source of the relationship is less obvious. The average spacingbetween coils is about 4.8 (A which means that the distances between the equivalent atoms areslightly longer than would give ideal packing of valines or isoleucines. However, in general each

Fig. 3. Stacking of non-polar residues in parallel b-helices. Carbons are drawn in light grey with oxygens and nitrogensin dark grey. (a) Aliphatic ‘‘cupped’’ stacks of isoleucines 212, 214, 240 and 242 and valines 265 and 267 on PB2 of the

polygalacturonase PehA. (b) The internal aromatic stack of residues phenylalanine 159 and 201 and the tyrosines 242,273 and 295 on PB3 of Bacillus subtilis pectate lyase. The tyrosine oxygens form hydrogen bonds with threonine 226,tryptophan 310 and two buried waters inside the parallel b-helix.

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sheet packs against at least one other so that small residues on, for example, PB1 and PB2 canpartially intercalate (see Section 1.2.5).

Although in general anti-parallel b-sheets do not allow stacking, an alternating stacking ofglutamines across an anti-parallel b-sheet, called polar zippers, has been proposed by Perutz et al.(1994) as a model for the interactions leading to aggregation between glutamine rich proteins.Such aggregates are involved in several neurodegenerative diseases such as Huntingdon’s disease.Only the w3 angles need to be moved from optimal values to give good hydrogen bonding andthere is good packing of the Cb and Cg atoms and thus some hydrophobic stabilisation of the b-sheet. Both asparagines and glutamines can form hydrogen bonds across an anti-parallel b-sheetbut asparagines in a parallel sheet can also form hydrogen bonds from Od1 to the NH of the otherasparagine, as well as the extra hydrogen bond of the T2 type turn (see Figs. 4 and 5). By contrast,no glutamine stacks have been reported in the experimental structures of parallel b-helicalproteins. Thus, a high ratio of asparagine to glutamine in a b-sheet protein may be suggestive ofuntwisted parallel b-structure and of the turns described below.

Fig. 4. Stereo views of T2 turns and asparagine ladders viewed from within parallel b-helices. Carbons are drawn inlight grey with oxygens and nitrogens in dark grey with some residue numbers indicted to the right of the a-carbons.

Hydrogen bonds of the central coils main chain and asparagine side chain are indicated by thin lines. The hydrogenbonding of PB2 (above) and PB3 (below) continues regularly. (a) Three coils of BsPel near T2 showing the first threeasparagines of the internal asparagine ladder. (b) Three coils of the T2 turn from RGase A showing continuous (if notregular) hydrogen bonding from PB2 to PB3.

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1.2.4. TurnsThe b-sheets of the right-handed parallel b-helical proteins often change direction sharply with

a single residue in aL-conformation interrupting the residues in b-conformation. To avoidconfusion it is important to distinguish between the angle through which the sheet is turned fromits extended direction and the interior angle (taking a single coil as a polygon). The chain turns 801at the T2 turn between PB2 and PB3 of the pectate lyases, giving an interior angle of 1001. Bycontrast, in a typical b-hairpin turn the chain changes direction by 1801. Many aL turns occur inthe right-handed parallel b-helix proteins but can be subdivided into at least two forms. The mostcommon form is shown in Fig. 4a. This was the form initially identified in the structure of PelC byYoder et al. (1993b). This turn involves two residues not forming all the possible main chainhydrogen bonds to the neighbouring coils. However, the simplest arrangement is that illustratedin Fig. 4b which occurs at the start of PB2 in BsPel and extensively in RGase A and thepolygalacturonases. The T2 turn of TSP also shows this regular hydrogen bonding towards thecarboxy-terminus of the parallel b-helix. This type of hydrogen bonding was identified as possible

Fig. 5. Asparagine ladders and T2 turns in leucine-rich repeat proteins. Carbons are drawn in light grey with oxygensand nitrogens in dark grey with some residue numbers indicted to the right of the a-carbons or carbonyl carbons.Hydrogen bonds from the main chain of the central coil and the central asparagine are indicated by thin lines. The

hydrogen bonding of the main sheet (PB2) above is regular but the residues below form less regular hydrogen bonds. (a)Stereo view of the asparagine ladder of insulin-like growth factor receptor domain 3. (b) Stereo view of the asparagineladder of U2A0. Only three of the four asparagines are shown.

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by Chothia and Murzin (1993). The turn shown in Fig. 4a can be derived from the simplerChothia and Murzin turn by rotating the amides of the residue after the a–L residue (denotedtherefore as residue i þ 1) inwards. At T2 in TSP at the amino terminal end of the parallel b-helixthese amides form hydrogen bonds with buried water molecules although the NH of 352 canhydrogen bond to Og of Ser 384. Similar structures also occur at T1 in TSP. In pertactin Ser 121and Ser 142, and in pectin methylesterase Ser 249 and Thr 294, can hydrogen bond to the inwardpointing NH from the previous coil. In the lyases a much more elaborate structure has evolved,the asparagine ladder (Fig. 4a) in which the main chain NH of residue i þ 1 hydrogen bonds to thecarbonyl of the asparagine side chain and the NH2 of the asparagine side chain hydrogen bonds tothe main chain carbonyl of the residue i � 2: Ribonuclease inhibitor shows a Cys–Asn ladderrelated to the asparagine ladder (Kobe and Deisenhofer, 1995b) but with the difference that theasparagines (and thus also the cysteines) only hydrogen bond to the main chain atoms. These havea peptide rotated away from b-conformation so that the i � 2 carbonyls can bond to theasparagine NH2 group rather than with the main chain from neighbouring coils. The humanspliceosomal protein U2A has ladders with only asparagines but follows ribonuclease inhibitor inthat the asparagine NH2 groups now bridge between each pair of i � 2 carbonyls, which are againdirected inwards (Fig. 5b). However, the insulin-like growth factor receptor (Garrett et al., 1998)shows asparagine ladders resembling those of the pectate lyases with main chain hydrogen bondsalong the ‘‘helix’’ axis.

The left-handed parallel b-helices also have aL-containing turns but their coils are much closerin shape to an equilateral triangle than are those of the righthanded parallel b-helices. Thus theyhave turns that give approximately 1201 changes of direction and 601 interior angles. These turnsoften form the 1–4 hydrogen bond of the classical b-turn but are clearly different from the latter asthe chains subsequently diverge. Interestingly, a similar turn also occurs in pertactin and Figs. 6aand b show that these turns are locally similar in parallel b-helices with the opposite hand.

The turns seen in pectate lyase were termed a distorted gbE turn by Yoder et al. (1993b) after theclassification of Wilmot and Thornton (1990). Chothia and Murzin (1993) viewed the turn as akink in a single continuous b-sheet, similar to the b-bulge (Richardson et al., 1978; Chan et al.,1993), whilst Pickersgill et al. (1994) identify the occurrence of aL-bounded b-strands as a newmotif. The turns in the left-handed parallel b-helices have generated less controversy, possiblybecause they were assumed to resemble those of the right-handed ‘‘family’’. A Wilmot andThornton definition for them might be bPgL; which is the classical type II turn of Richardson(1981) rather than bEgL: However, this turn also involves four residues rather than two outside thenormal b-sheet conformation and only one of the three peptides between them can form hydrogenbonds along the axis of the parallel b-helix.

So far turns involving an external aR residue have only been found in pertactin (Fig. 7a) butturns in which the aR residue is inside the parallel b-helix (and the surface is therefore concave)occur in all the right-handed parallel b-helix enzymes at the start of PB1 (Fig. 7b). These can alsoform hydrogen bonds along the axis of the parallel b-helix. The groove formed by this turn isnormally part of the active site of these enzymes.

1.2.5. Packing of b-sheetsThe packing of two b-sheets in a sandwich is a common feature of many protein folds and has

been extensively studied Chothia et al. (1997). There is a correlation between the optimal relative

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orientation of the b-sheets and their twist, with low twist allowing the sheets to pack with theirstrands (anti)parallel. Most b-sandwich proteins have more twisted sheets and the strands arerotated by an angle of about 301, or pack approximately orthogonally. In the right-handedparallel b-helices, PB1 and PB2 form such a sandwich in RGase A and the polygalacturonases.

Fig. 6. Stereo views of LpxA (UDP-N-acetylglucosamine acyltransferase from E. coli) together with P69 pertactin aftersuperposing all the main chain atoms of residues 116–123 of LpxA on residues 352–359 of pertactin with an RMSD of0.78 (A. The side chains pointing out of the parallel b-helix towards the viewer are reduced to alanines for clarity. (a)

Stereo view of the bonds of LpxA drawn as cylinders. Carbons are drawn in light grey with oxygens and nitrogens indarker grey. Some LpxA residues are numbered. (b) Stereo view of the bonds of LpxA around residues 121 drawn asthick grey lines together with the bonds of pertactin drawn as thinner black lines. Some pertactin residues are

numbered. (c) Stereo view of the bonds of pertactin drawn as cylinders. Carbons are drawn in light grey with oxygensand nitrogens in darker grey. Some pertactin residues are numbered.

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The helix becomes more triangular in shape in the other proteins and especially in pertactin andpectin methylesterase, while the left-handed parallel b-helices are close to an equilateral triangle incross-section. In RGase A and the polygalacturonases, PB1 and PB2 are rather flat and packalmost anti-parallel. When viewed down the helix axis, the g-carbons from PB1 and PB2 arenearly aligned so that they approach each other rather than packing as knobs into holes.However, this sandwich is unusual in being formed from a purely parallel sheet. The g-carbons areslightly off the perpendicular to the helix axis with those of PB1 directed slightly towards theC-terminus while those of PB2 are directed towards the N-terminus. This allows opposingresidues to avoid steric clashes between the sheets by simply having w1 near 1801 which also avoidsclashes within the sheet. The g-carbons from both sheets are generally directed towards the N-terminus. However, there is no steric clash because the b-carbons from PB1 of one coil slotbetween the g-carbons from PB2 of the same coil and the equivalent residue in the next coil. Thusin this case the local preference for w1; ‘‘cupped stacking’’, the rather flat b-sheets, and the optimal

Fig. 7. Stereo views of right-handed a-helical residues forming turns in the right-handed parallel b-helix family.Carbons are drawn in light grey with oxygens, nitrogens and a sulphur in darker grey. (a) The turn from T3 into PB1 at

the active site of the polygalacturonase PehA viewed from along PB1. The active site residues Asp 202 and Asp 224 areshown but Asp 205 is omitted. (b) Part of the stack of a-helical residues at T3 near the C-terminal end of the pertactinparallel b-helix. Hydrogen bonds of the main chain are indicated by thin lines. The hydrogen bonding is not completelyregular with two protons being donated to the carbonyl of Asn 442 (note that the residue numbers are to the left of the

residues in this image only).

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anti-parallel packing combine to stabilise the right-handed parallel b-helix architecture. However,the other, more triangular structures are also stabilised by the packing of stacked residues fromtwo or three b-sheets. The b-roll protease structures (Section 2.9) all show almost exact anti-parallel packing but the sheets pack unusually close together so that the residues mustinterdigitate unless one is a glycine. Thus the pleating in these b-sheets is anti-correlated betweenthe sheets. In the thicker b-roll like section of pertactin (Section 2.4) the packing involves atryptophan from each coil packing against two leucines from the other sheet. The right-handedantifreeze protein from Tenebrid molitor has similar spacing to the b-roll structures with cysteineson opposite sides forming disulphide bridges but only two adjacent sides of the helix are b-sheetand thus only one of the opposed cysteines comes from a b-sheet.

2. Description of known structures

2.1. Pectinases

2.1.1. The extra-cellular pectate lyase familyThe family contains most known pectate lyases, including all those with published three-

dimensional structures starting with the archetype PelC (Yoder et al., 1993a), and all knownpectin lyases (Henrissat et al., 1995). Henrissat and Coutinho have developed a web site,, displaying lists of polysaccharide lyases classified intohomologous families where this family is listed as family 1.

The X-ray structures of five members of this family have been published (see Table 1) andcrystals of one more have been reported (Doan et al., 2000). The known structures comprise threerather distantly related pectate lyases, PelC and PelE from Erwinia chrysanthemi and the BacillusSubtilis pectate lyase, and the two pectin lyases A and B from Aspergillus niger, PnlA and PnlB,which are more closely related with more than 60% sequence identity. The overall fold of thesestructures is shown in Fig. 8. There is a structurally conserved core comprising the parallel b-helix,the N-terminal helix and the N- and C-terminal extensions. The shape of the parallel b-helix isformed by three b-sheets and is very similar in all five enzymes. It is often described as L shaped,which arises because strands PB2 and PB3 are relatively long and make a slightly obtuse interiorangle at T2 (about 1001) and PB1 is often preceded by a residue in (a–R conformation and thenruns roughly antiparallel to PB2 (Figs. 1 and 7b).

All five structures are similar in the general pattern of long and short T1 and T3 loopsespecially in having long T3 loops in the coils to the N-terminal end of the parallel b-helix.These T3 loops are sometimes long enough to bury some hydrophobic residues as well as forminghydrogen bonds. Thus this region sometimes appears to form a non-contiguous ‘‘domain’’.However, there is no information to suggest that any of these structures can fold independentlyof the parallel b-helix. It is also worth noting that there is little similarity in the detailed structuresof the T3 and T1 loops between these enzymes except between the two pectin lyases. For example,although each of the pectate lyases has a helix in its first T3 loop, this coil in PelC does nothave a PB3 but after a few residues in irregular conformation starts an a-helix which packsagainst PB3 of the next two coils, while PelE and BsPel have a PB3 and thus leave the coilat a different angle. The T3 loop is much shorter in PelE (residues 58–74) than in BsPel

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(residues 64–121) where the loop continues out through irregular structure and two turns of a-helix (residues 85–91) before returning as a long helix (residues 104–121) which overlaps with ashorter helix in PelE for the last 3 turns (residues 67–74). The pectin lyases have long loops whichroughly but not exactly overlap the loop of BsPel but consist of irregular structure and a long b-hairpin.

The T1 regions include both extended irregular structure and shorter chains in mostly extendedconformation resembling short b-strands as part of the parallel b-helix, which, however, do notform all the hydrogen bonds needed for the region to be classified as a b-sheet. In fact the lyases

Fig. 8. Comparison of the pectinase folds, showing that even the obviously homologous lyases have very different loop

regions. Bacillus subtilis pectate lyases with a single bound calcium at (b), Aspergillus niger pectin lyase A at (d), Erwiniachrysanthemi pectin methylesterase, PemA, at (a) and the Erwinia carotivora polygalacturonase PehA at (c) are shown.The three common b-sheets PB1, PB2 and PB3 are coloured yellow, green and red, while the extra sheet PB1a of the

polygalacturonase family is dark blue. The b-hairpins in the T1 region of pectin methylesterase are cyan. All the activesites are towards the viewer and the N-terminal ends of the parallel b-helices are at the top.

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are intermediate between the esterases where this region is definitely not a b-sheet and thehydrolyases where it forms PB1a as shown in Fig. 2.

Jenkins et al. (1998) compared the lyases except for PnlB and determined that PelE and BsPelhave 31–34% identical residues after structural alignment (depending on whether alignment wasautomatic or assisted by human intervention), while the other pairs had between 13.6% and 17%identical residues after structural alignment (PnlA was most different from the pectate lyases).This low level of sequence identity allows us to have complete confidence that these arehomologues but makes alignment difficult without knowledge of the structure. For example,Henrissat et al. (1995) used the three pectate lyase structures to align all the then known sequencesbut chose not to align several regions of the fungal pectin lyase sequences, including one active siteT3 region, because there was insufficient similarity. Examination of the aligned sequences suggeststhat there may also be a bacterial pectin lyase family, which has diverged as far from the pectatelyase and the fungal pectin lyases as these have from each other. Pissavin et al. (1998) recentlyconvincingly suggested that PelZ from Erwinia chrysanthemi is another even more remotehomologue.

The structurally aligned primary structures show rather few identical residues overall but thereare two clusters of conserved residues. One represents the active site as expected but the second,including the sequence W(I/V)DH, is on the opposite side of the parallel b-helix from the activesite near T2 of the coils bearing the active site residues. This second cluster does not seem to beassociated with enzymic activity but may be critical for folding or stability. This region is at leastpartially buried by the N-terminal extension and the C-terminus is also nearby. The X-raystructures tend to show low temperature factors for that region and Jurnak et al. (1996) foundthat mutation in that region impaired folding. The conservation of some other residues, such asthe asparagine ladder, shown in Fig. 4, is more simply explained. Some other conserved residuesare involved in the folding of the N-terminal extension, while one asparagine forms a hydrogenbond to the main chain of the C-terminal region. Kamen et al. (2000) have studied thedenaturation and renaturation of PelC and find that the protein may unfold in two structuralblocks, but these could not be clearly identified in the structure. The unfolding transition is highlyco-operative and the protein was described as unusually stable.

The active sites of the pectate and pectin lyases are very different with mostly small hydrophilicside chains forming the pectate lyase active site and many large aromatic side chains dominatingthat of pectin lyase. As expected pectate lyase has a positive potential while pectin lyase isnegatively charged. Calcium is essential for pectate lyase activity but its precise role has beenpoorly understood until very recently. The structure of the calcium complex of Bspel (Pickersgillet al., 1994) showed that there was a single high affinity site and its affinity could also be measuredcalorimetrically as 0.2mM. However, the kinetic constant Km for calcium was much higher (near1mM) suggesting that calcium had a more complex role (Smith, D., unpublished). Scavetta et al.(1999) overcame many difficulties to determine the structure of a complex of the R218 K mutantof PelC with calcium and the substrate pentagalacturonic acid. The surprising result was that fourcalcium sites were found including one equivalent to that seen in BsPel. All the calcium atoms hadat least one atom from both the protein and from the substrate as ligands and thus formedbridges. Only four galacturonates were observed implying either that the substrate had beenpartially degraded or the extra site was either blocked or energetically unfavourable for binding.The penta-(or tetra-)galacturonate bound almost parallel to the parallel b-helix along the grove in

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PB1 interacting with ligands from T3, PB1 and T1. Not all the calcium ligands were conservedeven between the three known structures of the pectate lyases but it is possible, for example, thatPelC’s Asp 160 and Asp 162 might be replaced by PelE’s Glu 124 or BsPel’s Asp 170 which comefrom different T3 loops to a similar position. Thus, it is likely that Scavetta et al. have solved thebasic problem of how the extra-cellular pectate lyases bind their substrates although it is probablethat a longer substrate would bind at extra sites and possibly use extra bridging calciums. Somedifferences in the substrate binding between the different pectate lyases are also suggested by thealterations in the side-chains binding the calciums. The structure of the complex immediatelyaccounted for most of the available data accumulated by site directed mutagenesis. Kita et al.(1996) had shown that mutation of Asp 131, Glu 166, Asp 170, Lys 190, Arg 218 and Arg 223 ledto reduced activity. Except for the already mutated Arg 218, all of these are ligands of eithercalcium or the substrate and are generally conserved except that Glu 166 is an aspartate in mostpectate lyases.

As there was no potential base near any of the C5 atoms of the substrate, Scavetta et al.proposed a mechanism assuming that the sites occupied in the complex were �3 to +1 and thatArg 218 would have the same position in the productive complex as it had in the free enzyme. Thiswould place the arginine’s NH2 2.6 (A from the C5 in an ideal position to act as the base removinga proton from C5 with the b-elimination at O4 occurring in either a stepwise or a concertedreaction. This explains the complete conservation of the arginine in the family and the dramaticloss of activity on its mutation but has not won universal acceptance (Huang et al., 1999). The useof an uncharged arginine as a base might be plausible if the pH optimum for activity were highand the arginine was close to one or more calciums. However, only a very small fraction of Arg236 of pectin lyase A would be uncharged at its pH optimum near 5.5 and no calcium ions arerequired for pectin lyase activity. The review by Herron et al. (2000) gives a detailed view of themechanism and its relation to pathogenesis by Erwinia.

The pectin lyases have a ‘‘simpler’’ active site than the pectate lyases, consistent with having asimpler task, as pectin will easily undergo non-enzymatic b-elimination. They have no knowncalcium binding, no lysine homologous with Lys 190 of PelC and only one of the threecarboxylates of the high-affinity calcium site of pectate lyase site is conserved. This carboxylate isthe one furthest from the substrate and appears to function to orient the arginine that hasreplaced Glu 166 of PelC. The pectin lyases A and B of Aspergillus do present a new problem inthat activity is lost above pH 7. This may be associated with the two different structuresreported by Mayans et al. (1997) at pH 6.5 and 8.5 of PnlA from two different strains ofAspergillus niger. At pH 6.5, Asp 186 and Asp 221 are buried within the parallel b-helix andform a hydrogen bond while at pH 8.5, Asp 186 has turned outwards and been replacedby Thr 183 with a significant rearrangement of the T1 region. This conformationalchange is probably associated with pH rather than with the small number of amino acidsubstitutions between the strains, differences in glycosylation or altered crystal packing.PnlB at pH 5.5 and high salt concentration (Vitali et al., 1998) has a very similar conforma-tion to the pH 6.5 structure of PnlA, crystallised from polyethylene glycol. However, it isnot clear how the conformational change is related to the loss of activity or its biologicalfunction. Jaap Visser (pers. comm.) has suggested that a pectin lyase is unnecessary above pH 7when pectin is labile and that PnlA may change conformation to accelerate its recycling byproteases.

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2.1.2. Polygalacturonases and rhamnogalacturonase AThe polygalacturonases and two rhamnogalacturonases were identified as family 28 of the

glycosyl hydrolyases (Henrissat and Bairoch, 1996) and have been shown to act with inversion ofanomeric configuration (Biely et al., 1996; Pitson et al., 1998). Three crystal structures ofrhamnogalacturonase A from Aspergillus aculeatus (Petersen et al., 1997), the endo-polygalactur-onase PehA from Erwinia carotivora (Pickersgill et al., 1998) and endopolygalacturonase II or PGII from Aspergillus niger (van Santen et al., 1999) have been determined and the crystallisation ofseveral further enzymes has been reported (Yoder and Schell, 1995; Federici et al., 1999; Lu et al.,2000). Although these three enzymes are clearly homologous, they resemble the lyases in that theyhave diverged beyond the level of sequence identity where accurate models can be constructed. Allwere solved by using heavy atom derivatives rather than molecular replacement (for example, vanSanten et al. (1999) report that superposition of PG II and PehA gives an rms deviation (RMSD)of 1.8 (A for 265 Ca atoms and 19% sequence identity). Despite the change of function, RGase Ais slightly more closely related to PG II than the two polygalacturonases are to each other and itmay thus have diverged from PG II after the fungi diverged from bacteria. In fact PehA is alsovery slightly closer to RGase A than to PG II and superposition by O (Jones et al., 1991) gives1.6 (A for 280 Ca atoms between RGase A and PG II, 1.67 (A for 253 Ca atoms between PehA andPG II, and 1.67 (A for 280 Ca atoms between PehA and RGase A (using chain B for PG II).

RGase A and PehA have N-terminal extensions which both start with the amino terminusforming hydrogen bonds to a T1a turn residue in a–L conformation. In PehA this residue is Asn264 and the OD1 also bonds to the N-terminus while in RGase A the N-terminal residue is anasparagine and forms an internal hydrogen bond so that only the carbonyl of Asp 235 bonds tothe amino terminus. Ser 3 also makes a conserved hydrogen bond to an a–L residues at T1a (Asn185 of RGase A or Asn 211 of PehA). The nearby residues lie in the same region with Leu 2 andVal 6 of RGase A packing in approximately the same position as Asp 2 and Arg 4 of PehA butforming very different interactions. The chains then diverge with Glu 8 of PehA forming a saltbridge to another a–L residue, Lys 189, which is at T2. At the equivalent position at T2 RGase Ahas Asn 165 which forms a hydrogen bond to the C-terminal extension of RGase A. RGase Aforms a short a-helix and Cys 15 of PehA and Thr 20 of RGase A come together again near thesecond residue of PG II, Ser 29 (in fact Asp 28 of PG II is close to Lys 19 of RGase A) and enterthe parallel b-helix.

The parallel b-helix of PehA ends abruptly with the final strand of PB1a, while that of PG IIloops back to form a C-terminal disulphide bridge which may cap the parallel b-helix. RGase Ahas a long C-terminal extension which forms a strand of anti-parallel b-sheet with the last strandof PB1 (residues 369–375) and then packs against PB3 burying several aromatic residues such asthe externally stacked pair of Tyr 215 and 242. It also partially buries the longer stack of Phe 104,His 141, Tyr 164 and His 189 from the end of PB2 by forming a S bend (residues 386–398–412–421) which lies against the PB3-T3 region. Thus the C-terminal extension like the N-terminalextension interacts with a stack of a–L turns (at T2 in this case). The main chain interactions withthe Asp 142, Asn 165 and Asp 190 may be especially significant. A final factor that may stabiliseRGase A is its glycosylation which is unusually well ordered in the crystal structure.

The first obvious distinguishing feature of the central parallel b-helix of the family 28 enzymes isthe occurrence of the fourth PB1a b-stand (PB2a) as shown in Fig. 2. In fact the conformationaldifferences in this region between a pectate lyase such as BsPel and the hydrolases are not as

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striking as might be imagined. RGase typically has two residues in b-conformation flanked by tworesidues in a–L conformation forming hydrogen bonds along the helix axis. The b-arch of BsPel atresidues 148–151, 190–193 and 231–234 has glycines at 190, 191 and 231, which together with thesmall Ser 149 and Ala 232 allows these residues to adopt an irregular conformation while the Capositions are close to the equivalent residues of RGase A. However, residues 150–151, 192–193and 233–234 of BsPel have essentially the same conformations and hydrogen bonds as theequivalent residues of RGase A.

The second feature is that the structure of RGase A is generally much more regular than that ofthe lyases and forms many of the hydrogen bonds needed to generate the ‘‘perfect’’ parallel b-heliximagined by Chothia and Murzin (1993) but not actually seen in the pectate lyases. For exampleat T1 Gly 70, Asp 97, Asp 134 and Thr 157 are all in the a–L conformation in successive coilsforming hydrogen bonds along the parallel b-helix. This is followed by a coil with a continuousPB1-PB1a b-sheet, and finally by three more distorted turns with one residue in a–L conformationbut without regular hydrogen bonding. At T1a these coils have Ala 73, Asp 100, His 137, Asp 160,Asn 185, Asn 208, Asp 235, Asn 263, Asn 299, and Asp 330 in a–L conformation forming regularhydrogen bonds along the parallel b-helix before ending with the more continuous sheet asdescribed in the introduction. Finally at T2 after two irregular turns Asp 142, Asn 165, Asp 190,Ser 213, Asn 240, Asn 268, Asn 304 and Asp 335 are in a–L conformation with the regularhydrogen bonding pattern. The differences between the type of a–L turn generally seen in thelyases and the hydrolases are illustrated in Figs. 4a and b. Some, but not all, of the asparagineresidues in these turns form regular external stacks. There is also often a residue in a–Rconformation at the start of PB1 and again RGase A shows regularity with Met 92, lle 129, His150, Gly 178 and Cys 199 in a–R conformation, followed by an irregular coil, then Met 249, Ser277 and Pro 317 in a–R conformation without making all the possible hydrogen bonds and finallya longer PB1 making anti-parallel sheet with the C-terminal extension.

PehA and PG II have very similar parallel b-helices and interior residues to RGase A. As well asslowing the evolution of the interior, the conservation of the overall shape of the coils may makecompensating mutations rather more probable than usual. For example, Met 249 in RGasebecomes Gly in PehA and Ala in PG II but Gly 271 of RGase A is replaced by Met both PehAand PG II to conserve the volume. The four disulphide bridges between Cys 21 and Cys 47, Cys199 and Cys 216, Cys 322 and 328, and Cys 350 and Cys 359 are conserved between RGase A andPG II (although the last does not have the same structure) but are not found in PehA, whichhowever has a single disulphide between Cys 89 and Cys 99 in a T3 loop. An unusual feature ofRGase A is the cluster of three cysteines 199, 216 and 222 where Cys 222 is in the positionequivalent to Cys 199 in the next coil but does not have the same a–R conformation. It is not clearif Cys 222 assists in the folding. The large cavity reported in RGase A seems to be mostly occupiedby Phe 74 in PG II and at least partially by Leu 65 of PehA.

The family 28 enzymes have very many aliphatic residues both ‘‘aligned’’ and forming ‘‘stacks’’and these dominate the interior of the parallel b-helices. Although there are several aromaticresidues, there are very few internal aromatic stacks: one of Phe 129, Phe 152 and Phe 182 in PG IIat the start of PB1, Phe 263 and Trp 305 at the asparagine ladder position of RGase A at the startof PB3 as shown in Fig. 4b (Phe 101 and Phe 138 are aligned) and Tyr 296 and Phe 330 on PB2 inPehA (Phe 162 and Phe 185 have similar conformations but do not interact strongly). There areno internal polar stacks in RGase A or PG II but there is a single Asn 245 at the asparagine ladder

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position of PehA. This hydrogen bonds to the main chain NH of Ser 217 in the previous coil. OGof Ser 217 in turn hydrogen bonds to the main chain NH of Thr 190 but OG1 of Thr 190 onlyhydrogen bonds weakly to the carbonyl oxygen of Leu 164. To the C-terminal side, ND2 of Asn245 hydrogen bonds to a buried water and to the carbonyl oxygen of Val 214. The buried water iscapable of bonding to three carbonyl oxygens of residues 242, 268 and 269 (as Asp 269 forms ana–L turn). The existence of a stable ‘‘asparagine step’’ suggests that the evolution of newasparagine ladders is possible.

As discussed in the introduction, the parallel b-helices of RGase A and PG II finish with adisulphide and may have a topological winding number of 13, while PehA finishes with the finalPB1 a and has a winding number of 12. All of the family 28 hydrolases have 10 coils as defined byYoder et al. (1993b). The pattern of long T3 loops especially at the N-terminal end of the parallelb-helix and long T1 loops towards the C-terminal end is also seen in these hydrolases. Both PehAand PG II have longer T3 and T1 loops than RGase A, so that the active sites become deeper andnarrower clefts. However, there is no detailed similarity in the loop conformations suggesting thatthis similarity reflects the requirements of polygalacturonase as opposed to rhamnogalacturonaseactivity. RGase A has several unusually well-ordered N-inked sugar residues at the C-terminalend of the parallel b-helix.

The active sites of the family 28 hydrolyases have not yet been fully identified by determiningthe structure of a complex with a substrate, product or inhibitor. However, sequence conservationand analogy with other enzymes suggests that substrates bind to the cleft formed by the T3–PB1–T1 region and that catalysis involves three carboxyl residues: Asp 177 (Asp 202, Asp 180), Asp197 (Asp 223, Asp 201) and Glu 198 (Asp 224, Asp 202), where the RGase A residue is givenfollowed by the PehA and PG II equivalent residue in brackets. Van Santen et al. (1999) reportedthat site directed mutation of PG II gave activities of 0.01% and 0.08% for D180E and D180N,0.01% and 0.01% for D201E and D201N, and 0.6% and 0.01% for D202E and D202Nwhile only slightly changing the Km values. The H223A mutant also had only 0.5% of the WTspecific activity and no change in Km but R256N and K258N showed 14% and 0.8% of theWT specific activity with an order of magnitude increase in Km. Histidine 223 of PG II(His 251 of PehA) is not conserved in RGase A but may thus have a critical role in binding thesubstrate in polygalacturonases in an altered conformation to assist the catalysis. Armand et al.(2000) report the properties of these mutants in more detail as well as mutations at Arg 256 andLys 258.

As this family of enzymes was known to catalyse hydrolysis with inversion of anomericconfiguration, both Pickersgill et al. (1998) and van Santen et al. (1999) noted conserved watersites in contact with two of the conserved carboxylates. Similarities to the active site of the P22phage tailspike endorhamnosidase were noted as well as the fact that the distances between thecarboxylates did not fit the rules used previously for identifying inverting and retainingglycosidases. However, these rules were developed for b-linked sugars such as the substrates ofxylanases and cellulases where the lone pair on the glycosidic oxygen which is protonated by theacid catalyst is on the opposite side of the sugar to the attacking nucleophile. The 7–8 (A distancefor these inverting family 28 hydrolases suggests that the lone pair is not pointed directly awayfrom the nucleophilic water in these enzyme substrate complexes. Van Santen et al. are moreexplicit in proposing the water in contact with and activated by Asp 180 and Asp 202 (water 2 ofPehA and water 37 of RGase A) as the nucleophile attaching at C1. Asp 201 of PG II is assumed

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to act as an acid to protonate O1 (i.e. the leaving group). This is plausible as Asp 202 is in contactwith the positively charged Arg 256 and is likely to be charged.

Pag"es et al. (2000) reported the properties of the mutants N186E and D282K, which impliedthat the substrate binds with its reducing end towards the C-terminus of the parallel b-helix as isseen in PelC by Scavetta et al. (1999) and that N186 was part of subsite �4 while D282 was part ofsubsite +2. Mutations were also reported of residues forming the more central subsites and theseoften showed greater effects on the overall activity. Examples include D183N at subsite �2, andY291L or Y291F at subsite +1. Finally the mutation E252A allowed PG II to accept amethylated substrate more easily.

As might be expected given the same substrate, the general nature of the residues at the activesites of these enzymes resembles those at the active sites of the pectate lyases. However, calcium isnot required for substrate binding or activity. Some aromatic residues are found such as Phe 151,Trp 182, Tyr 276 and Trp 284 of RGase A, Phe 175 and Tyr 231 of PehA and Tyr 130, Tyr 254,Tyr 283 and Tyr 326 of PG II (as the exact binding site is unknown we cannot assert that all theseresidues are in contact with the substrate in a productive complex). None of these aromatic sidechains are conserved in all three structures.

2.1.3. Pectin methylesteraseThe structure of the pectin methylesterase PemA from Erwinia chrysanthemi has been reported

by Jenkins et al. (2001) at 2.4 (A resolution. It has the same number of coils as the pectate andpectin lyases and the coils of this right-handed parallel b-helix have the same general shape. PemAhas an a-helix at the N-terminal end of the parallel b-helix, long T3 loops and long T1 loopstowards the C-terminal end of the parallel b-helix, and a C-terminal extension. The C-terminalextension interacts with PB2 rather than PB3 as seen in the lyases and is longer than that of thelyases. It also makes interactions with the parallel b-helix in the region which interacts with the N-terminal extensions of the lyases and the hydrolyases.

In terms of the shape of the coils, the structure represents a divergence from the lyases in theopposite direction from the hydrolyases with little trace of the PB1a sheet and a short T1 archregion. Stacked a–R residues are again found at the start of PB1 but the angle between PB2 andPB3 is less obtuse than in the other pectinases so that the distance between PB3 and the start ofPB1 is shorter. This arises because there is no internal stack of aromatic residues on PB3 as seen inthe lyases. The overall shape of the coil is similar to that at the N-terminal end of the parallel b-helix of pertactin (see Section 2.4).

PemA shows many internal aliphatic stacks and an internal aromatic stack, which occur onPB2. An external asparagine stack is found but the asparagine ladder position of the lyases isfrequently a cysteine in the pectin methylesterases. In PemA the disulphide between Cys 192 andCys 212 appears to be partially formed but the crystals had been treated with the reducing agentDTT as 100mM DTT did not inhibit the activity (K. Worboys, unpublished observations). It isnot clear if the cysteines in PemA and in its homologues generally form disulphides or stacks ofcysteines although some sequences contain cysteines which cannot form disulphides.

There is a deep cleft along the parallel b-helix formed the T3–PB1–T1 region which contains themost conserved sequences and corresponds to the substrate binding site of the lyases. The T3 andT1 loops of PemA have no detailed similarity to those of the lyases and the cleft is rather deeper(as is the active site cleft of the polygalacturonases). Two of the T1 loops form b-hairpins and with

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one hydrogen bond between the hairpins almost form a four stranded anti-parallel b-sheet. Thecleft resembles the pectin lyases in having many aromatic residues including an external aromaticstack of Tyr 158, Tyr 181 and Phe 202 on PB1. The similarity between pectin methylesterases andpectin lyases results from convergent evolution to bind the same substrate because only onearomatic residue, Phe 202 of PemA and Tyr 215 of PnlA, occurs at an equivalent position.

Only the sequence conservation (Markovic and Jornvall, 1992; Laurent et al., 1993) and theobserved pH activity profile gives us any information on the catalytic mechanism of PemA. Theenzyme has a broad roughly bell-shaped pH optimum from about pH 5–9 (Pitk.anen et al., 1992).However, if the active site has been correctly identified, the only conserved potentially catalyticresidues are Asp 178, Asp 199 and Arg 267, and the last two form an ion pair. As Asp 199 is likelyto be deprotonated, only Asp 178 is likely to act as an acid and either Asp 178 after it has donateda proton or Asp 199 or both may activate a water molecule to act as a nucleophile. PemA is thusthe first member of a new class of aspartyl esterases.

2.1.4. PelL from Erwinia chrysanthemiThe structure of PelL from Erwinia chrysanthemi is currently being determined in our

laboratories. This is a member of family 9 in Henrissat and Coutinho’s classification of thepolysaccharide lyases. Although the sequences are unrelated (Lojkowska et al., 1995), PelLresembles the ‘‘extra-cellular’’ pectate lyases in requiring calcium for activity. PelL is an endo-pectate lyase and makes a significant contribution to tissue maceration in vivo. However, itsexpression in Erwinia is not regulated by the same mechanism that controls expression of the‘‘extra-cellular family’’ enzymes. Crystallographic refinement has not yet been completed but thecurrent model with an overall R factor below 17% and a free R factor below 19% to 1.6 (Aresolution allows almost all the residues to be clearly defined.

The architecture of PelL is a right-handed parallel b-helix and the overall shape of the coils issimilar to that of the extra-cellular lyases with three b-sheets, PB1, PB2 and PB3. The T1 regionresembles BsPel, generally with an a–L turn at the start of PB2 but without a regular PB1 a sheetas seen in the polygalacturonases and rhamnogalacturonase A. However, there are 10 coils as inpolygalacturonase rather than eight, as in the other lyases. There is an N-terminal a-helix as in allthe right-handed parallel b-helix proteins except pertactin (Emsley et al., 1996). There is a shortN-terminal extension of residues 26–39, numbering residues from the gene so that Ala 26 is thefirst residue of the mature enzyme, and a long C-terminal extension of residues 357–425 whichpacks against PB3. PelL has 12% of asparagine in its sequence and as initially suggested byLojkowska et al. (1995), these form both internal and external stacks. However, the internalasparagine ladders occur at the start of PB2 (T1a) and at T3 rather than at T2. There is a clusterof conserved residues including Asp 209, Asp 233, Asp 234, Asp 237 and Lys 273 in the T3–PB1–T3 region where all the known pectinase active sites are located and this region can be tentativelyidentified as the active site of PelL.

2.1.5. Pectate lyase Pel-15 from Bacillus sp. strain KSM-P15Akita et al. (2000) have reported the crystallization of a pectate lyase Pel-15 from Bacillus sp.

strain KSM-P15 with a very alkaline pH optimum of 10.5 and although no article has yet beenpublished describing the structure, the coordinates have been deposited to the Protein Data Bankas accession 1EE6. This structure is the first for a member of the family 3 lyases according to

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Henrissat and Coutinho’s classification. The structure is a right-handed parallel b-helix with asimilar shape for each coil to that of the extra-cellular lyases and also has 8 coils together with anextra PB3 strand. However, the sequence is significantly shorter than that of the other pectinasesand there is no N-terminal helix, there is only one notable T3 loop (residues 27–42) and there is noC-terminal extension. The small size is not associated with an unusually repetitive structure. Thereare left-handed a-helical turns and some stacking of aliphatic groups. However, the onlyasparagines stack is external and there are no obvious aromatic stacks. Alignment with Bacillussubtilis pectate lyase gives an RMSD of 1.94 (A for 158 Cas (of 197), showing that this enzyme isanother member of the pectinase superfamily. The active site contains several striking similaritiesto the other lyases and some surprises, including the site of calcium binding. The calcium positionis different from that of the single calcium seen in BsPel and PelL. This site also does notcorrespond to any of the sites reported by Scavetta et al. (1999) for PelC but is near aspartates inboth PelE and BsPel. A full description of this interesting structure is eagerly awaited andhopefully it will be possible to model or observe substrate binding and thus understand in detailthe role of calcium, which is also necessary for the pectate lyase activity of this family.

2.2. The P22 phage tailspike endorhamnosidase

The P22 phage tailspike endorhamnosidase (TSP) has been for many years one of the principalmodel systems used to study protein folding and misfolding both in vivo and in vitro (King et al.,1996; Betts et al., 1997; Seckler, 1998; Betts and King, 1999). The final rate-limiting trimermaturation reaction, which has the same rate in vivo (Goldenberg and King, 1982) and in vitro(Danner et al., 1993), produces a folded trimeric enzyme which is unusually stable both to hightemperatures and even more usefully in presence of denaturants including SDS. Thus this systemhas allowed a clear separation of issues relating to folding from those relating to the stability ofthe folded protein. Many temperature sensitive folding (tsf ) mutants have been found which arestable once folded but which will not fold above some non-permissive temperature. Severalsupressor mutations (su) that restore folding to tpf mutants at the non-permissive temperaturehave also been identified. The determination of the TSP structure depended on the fragmentationof the enzyme by recombinant expression and crystallisation of the two fragments, residues 1–124and 109–666 (Miller, 1995; Miller et al., 1998a). The structures of these fragments of TSP(Steinbacher et al., 1994, 1997) revealed that the major part of the protein forms a parallel b-helixwhile the N-terminal (residues 1–108) and both C-terminal (residues 541–666) domains have anti-parallel b-folds. This information opened the way for the genetic and in vitro folding studies to beinterpreted in terms of atomic interactions.

The nomenclature describing the structure of TSP was developed independently from that ofthe pectinases. The articles describing the structure of TSP describe the three stranded parallelb-helix in terms of sheets A, B and C corresponding to PB2, PB3 and PB1 of the pectinases. Theoverall shape of the monomer resembles a fish with the parallel b-helix domain taken as the bodyand the extreme C-terminal domain taken as the ‘‘Caudal fin’’. The long loops off the parallelb-helix are then named ‘‘Dorsal and Ventral fins’’, corresponding to T3 and T1 loops,respectively. There are 13 complete coils in the parallel b-helix domain of TSP and these coils havethe characteristic L or kidney shape also seen in the pectinases and the N-terminal region ofpertactin with a stack of a–R residues at the start of sheet C (PB1). Like the pectinases but unlike

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pertactin, the parallel b-helix has an N-terminal a-helix instead of a PB1 in its first coil followed bythe first strand of sheet A (PB2). The detailed shape of the parallel b-helix is more similar to thepectic lyases than pectin methylesterase or pertactin, mostly because the a–R residue at the start ofsheet C (PB1) and the second inward pointing residue of sheet B (PB3) tends to be larger, forcingsheets B and C (PB3 and PB1) apart. One possible alignment of the parallel b-helix with BsPelgives an RMSD of 2.02 (A for 174 Ca atoms. There is one long T3 loop from residues 197 to 260forming the Dorsal fin. There are some long T1 loops, forming the Ventral fin, one of which ispoorly ordered in the X-ray structure. Although TSP is a hydrolyase, the T1 region is more similarto the pectic lyases than the hydrolyases and there is only a very small region where the hydrogenbonds of a sheet PB1a are present between residues 267–268 and 287–288. However, the T1 regionhas an interesting example of internal stacked cysteines. The turn at T2 is generally similar to thatseen in the pectic lyases and has similar geometry. The interior of the parallel b-helix is almostexclusively hydrophobic, without polar stacks such as the asparagine ladders of the pectic lyases,and with few buried waters, mostly associated with the amides of the T2 turn. There are severalaliphatic stacks especially at the C-terminal end of PB3 and an aromatic stack on PB1 (residuesPhe 284, Phe 308 and Phe 336). However, there are also aligned rather than stacked interactionsand edge to face aromatic interactions.

The fragment 109–666 had the same oligosaccharide binding and endoglycosidase activity asthe full length protein (Miller et al., 1998a) which acts both as an adhesion factor in phage bindingand as an enzyme. Steinbacher et al. (1996) determined the structure of three complexes of TSPwith receptor lipopolysaccharide fragments comprising two O-antigenic repeating units fromthree Salmonella species, giving the first direct evidence for the substrate binding site of a parallelb-helix enzyme. The substrate bound to the same cleft created by sheet C and the loops on eitherside (i.e. T3–PB1–T1) and was bound almost parallel to the helix axis as was later seen in pectatelyase C (Scavetta et al., 1999) and chrondroitinase B (Huang et al., 1999). The binding site shows‘‘wobble’’ with alternative binding sites for fragments from different strains but the terminalrhamnose is in almost the same position in the three complexes. The binding of the O-antigeninvolves mostly small hydrophilic side-chains and only the side chain of Lys 302 shows significantdisplacement on forming the complex. However, the binding does bury a considerable surfacearea, showing the excellent complimentarity of the protein and the polysaccharide. The reducingend of the polysaccharide was bound towards the C-terminus of the parallel b-helix. Aspartates392 and 395 and glutamate 359 were identified as likely to be involved in catalysis. Mutation ofany of these residues to the amide, either asparagine or glutamine, caused a dramatic loss ofactivity (Baxa et al., 1996) but all three mutants continued to bind substrate and product withwild-type affinity. An inverting endorhamnosidase mechanism was suggested (Steinbacher et al.,1996) in which a water molecule observed to bind in contact with Glu 359 and Asp 395 acts as thenucleophile, while Asp 392 protonates the glycosidic oxygen.

The structural data revealed that all the 62 tsf mutants were in the parallel b-helix region ofTSP (Haase-Pettingell and King, 1997). Miller et al. (1998a) had reported that the removal of theN-terminal domain to give the fragment 109–666 unmasked the effects of two tsf mutants, G244Rand D238S, and four suppressor mutants, V331G, V331 A, A334V and A3341, on the kinetics offolding and unfolding. Subsequently Miller et al. (1998b) reported the properties of a tailspikefragment corresponding to the isolated parallel b-helix domain consisting of residues 109–544(bhx). This fragment has low but measurable endorhamnosidase activity (0.2% of the wild-type)

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and is a mixture of monomers and trimers. Unlike the larger species, it folds and unfoldsreversibly. Further truncation at the C-terminus of the parallel b-helix produced increasinglyunstable proteins which could not be purified showing the importance of capping the parallel b-helix. Schuler and Seckler (1998) conclude that the P22 tailspike folding mutants act by globallystabilising or destabilising thermolabile monomeric folding intermediates in which the centralparallel b-helix is topologically similar to the native structure but less tightly packed. Betts andKing (1999) point out the alternative that the rate of the self aggregation process is reduced. Anobvious question might be why are the su mutant sequences not selected by evolution? Val 331 isclose to the substrate and the V331G or V331A mutants are less active than wildtype. The case ofA334V and A3341 is more complex in that the intermediate and bhx are stabilised by thesemutations, which produce a classical aliphatic stack, but the final trimer is destabilised (Beissingeret al., 1995) although it has a similar structure to the wild type enzyme (Baxa et al., 1999).

Recently Schuler et al. (2000) have described the rational design of mutants aimed at producingthe tsf and su phenotypes. A tsf mutant was produced by the mutation T326F, which introduced amuch larger residue into the parallel b-helix. The most surprising feature was how little thestructure was distorted with very small shifts of the main chain and only a slight but significantincrease in the dissociation constant for an octasaccharide (no change was seen with T326S andT326V). The steric strain was mostly absorbed in an unusual conformation of Phe 352 althoughstrain was transmitted as far as Val 362 on the opposite side of the helix. The rigidity of theparallel b-helix (or the plasticity of its internal residues) may suggest the mechanism which haspreserved the shape of parallel b-helices while erasing all trace of sequence similarity. Potential sumutants were designed by mutation of residues Glu 359 and Trp 391 at the active site followingthe hypothesis that these mutants had not been observed because of the loss of activity. Theseresidues’ conformation lies on the edge of the allowed region of the Ramachandran plot.However, E359A was much less stable than wild-type and E359G was still less stable than wild-type, suggesting that the side chain of Glu 359 is important for stability as well as for activity.W391A and W391G were also less stable than wild type. The binding of octasaccharide was alsoseriously affected but the crystal structures of E359G and W391A were similar to wild type exceptfor some small changes near the side chain of Trp 391.

After describing the role of the parallel b-helix in folding, it is necessary for balance to note thatRobinson and King (1997) showed that the formation and breaking of disulphides involving Cys613 and Cys 635 in the C-terminal ‘‘Caudal fin’’ domain was critical for the folding to the nativeconformation. This domain is formed by chains from each monomer of the trimer and has beencalled a triple b-helix by Seckler (1998). This domain contains anti-parallel b-sheet but Kreisburget al. (2000) note some similarity in the packing compared to the parallel b-helix and suggest thatthis packing might be a model for amyloid.

Misfolding of TSP has been studied by King and colleagues (King et al., 1996; Speed et al.,1996, 1997) with the surprising result that aggregation is specific rather than random and thatfolding intermediates do not coaggregate with each other but only with themselves. It was possibleto directly identify by native gel electrophoresis sequential multimers as the earliest intermediatesalong the in vitro aggregation pathway. Schuler et al. (1999) have similarly shown that bhxaggregates via a linear polymerisation mechanism observing monomers, dimers, trimers andtetramers. The secondary structure was relatively unchanged but tryptophan fluorescence wasquenched. Fibrils were observed which bound Congo Red and gave the green birefringence

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characteristic of amyloid. Foguel et al. (1999) used hydrostatic pressure to rescue native TSP fromaggregated material and suggest that the dissociation of the misfolded aggregates may be similarto the pressure induced dissociation of oligomeric proteins rather than the behaviour expectedfrom a random aggregate.

2.3. Chrondroltinase B from flavobacterium hepinarum

The structure of chondroitinase B from Flavobacterium hepinarum, which degrades dermatansulfate, has recently been determined at 1.7 (A resolution (Huang et al., 1999). It is a lyase formingan unsaturated 4–5 carbon–carbon bond at the non-reducing end of the product but unlike thepectin and pectate lyases it catalyses the cleavage of a b-(1–4) glycosyl bond. The mature enzymeis produced by removing a 25 amino acid leader peptide from the 506 amino acid precursor (theresidues numbering used below is that of the precursor). This structure is a right-handed parallelb-helix with 13 coils, making it the longest of the parallel b-helix enzymes. There is no obviousrelationship between the sequence of chondroitinase B and any other protein whose structure isknown. However, there is clear homology to two poly-b-d-mannuronate lyases fromPseudomonas sp. (Maki et al., 1993) and Photobacterium sp. (Malissard et al., 1993) and theselyases constitute family 2 of the alginate lyases as defined by Chavagnat et al. (1996) and family 6of Henrissat and Coutinho’s polysaccharide lyases.

The overall fold is similar to the other right-handed parallel b-helix enzymes both in the generalL-shape of the three sheets and in several specific features. Thus the parallel b-helix starts with ana-helix, finishes with a long C-terminal extension from residue 421 to 506 and including two a-helices (residues 452–458 and 491–503) which interacts with the PB2–T2–PB3 region. The parallelb-helix also has only the two residue gbE turn (Yoder et al., 1993b) at T2 but has long T3 loops,especially to the N-terminal side of the active site and long T1 loops to the C-terminal side of theactive site. However, chondroitinase B differs from the other right-handed parallel b-helixenzymes in lacking a large N-terminal extension and having an a-helix (residues 360–371) insertedin a very extended T1 turn in coil 11 which forms part of the active site. The final a-helix (residues491–503) is also unusual in interacting with the T3 loops forming the other side of the active sitecleft.

There are many examples of both stacking and alignment of similar residues in thechondroitinase B structure. The interior of the parallel b-helix contains aliphatic, aromatic andpolar stacks, including a very long stack of Phe 83, Phe 103, Phe 135, Phe 167, Phe 205, Phe 237,Tyr 261, Tyr 283, Phe 309, Phe 347, and Phe 388 on PB3. There is a classical asparagine ladder(together with cysteines) two residues before at the T2 turns from Cys 133, Cys 165, Cys 203, Asn235, Asn 259, Asn 281, Asn 307, Asn 345 and Asn 386. There are also external aromatic stacks.However, there are at least two aromatic clusters (Tyr 256–Phe 270–His 302 and Phe 357–Phe388–Phe 399–Phe 404–Trp 411) showing that the hydrophobic core of the parallel b-helix is notexclusively built from stacking interactions although stacked residues participate in the clusters.

The structure forms a L shaped cleft from the T3–PB1–T1 region to which Huang et al. (1999)bound a dermatan disaccharide product and determined the 1.7 (A resolution structure of thecomplex. There are some aromatic side chains lining the cleft (His 116, Tyr 222, Phe 296, Trp 298and Tyr 324). However, the most obvious interactions of the disaccharide are with polar andcharged residues (Asn 213, Glu 243, Glu 245, Lys 250, Asn 269, Arg 271, Arg 318, His 334, Arg

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363 and Arg 384), while His 116, Arg 184 and Glu 333 might interact with a substrate rather thana disaccharide. The only side chains moving significantly on forming the complex belong to Asn213 and Arg 363 (4 and 1 (A, respectively). Of the residues near the active site, conservationbetween the three sequences suggests that Lys 250, Arg 271 and Glu 333 may have important rolesin catalysis while Arg 318 and Arg 363 may be significant in defining the specificity.

The most surprising feature of the complex is that the disaccharide has bound with the reducingend towards the C-terminus of the parallel b-helix, in the reverse direction to that seen in thecomplexes of pectate lyase (Scavetta et al., 1999) and the phage P22 tailspike endorhamnosidase(Steinbacher et al., 1996). However, we should expect the mechanism of a lyase acting on a b-(1–4)bond to be very different from one acting on an a-(1–4) bond. Huang et al. (1999) argue that themechanism should be a two-step process of proton abstraction followed by b-elimination assuggested by Gacesa (1987), Gerlt and Gassman (1992) and Guthrie and Kluger (1993) ratherthan the concerted mechanism likely in the pectate lyases.

2.4. P69 pertactin from Bordetella pertussis

The structure of P69 pertactin (referred to as pertactin below) was determined at 2.5 (Aresolution by Emsley et al. (1996) and has the longest parallel b-helix observed so far with 16 coils.Pertactin is the amino-terminal external domain of a 93 kDa precursor, P93 pertactin, encoded bythe Bordetella pertussis gene prn. It is a surface-exposed domain of an outer membrane protein ofB. pertussis and is a component of some acellular whooping-cough vaccines. It has been identifiedas a virulence factor and a role in adhesion to mammalian target cells proposed (Leininger et al.,1991). However, the mechanism and importance of pertactins role in adhesion remains open(Everest et al., 1996; van den Berg et al., 1999).

Whilst the N-terminal region of pertactin does not start with an a-helix, it is otherwise rathersimilar in shape to the parallel b-helix enzymes. The shape and size of each coil of the parallelb-helix is similar and there are detailed features which enable the sheets PB1, PB2 and PB3to be aligned unambiguously. The T2 turns resemble those in the lyases, pectin methylesteraseor TSP rather than the family 28 hydrolyases. However, pertactin is possibly the mostregular of the parallel b-helical proteins with spectacular internal aliphatic stacks on ratherflat b-sheets. Examples include I17–I45–V79–L107–V120–L149–V186–V209–V264–I287–I310–L341–I363, I35–V61–L93–L123–I144–V174–I201–I224–V278–L302, L54–L86–A116–V137–V167–L194–V217–V271 V295–I327–L351–V381–L399–L417–L444, V81–V109–I131–I151–Vl88–A211–V266–V289–T312 and I118–V139–L169–L196–L219–L273–V297–L329–L353–L383–I401–L419. However, not all of these are real ‘‘stacks’’ as opposed to ‘‘aligned’’ residues, so that L196and L219 are only aligned and some alanines are included. Leucines can partially join the cuppedstacking of valines and isoleucines but do introduce some irregularity.

The shape of the substrate binding sites of the parallel b-helical enzymes is maintained with afeatures such the stack residues in a–R conformation at the start of PB1. No known activity isassociated with this region in pertactin and the RDG (226–228) sequence which may bebiologically important (Everest et al., 1996; van den Berg et al., 1999) is in the PB3-T3 region atthe start of a very long loop which folds over the region equivalent to the active sites of theenzymes. Most of the pertactin residues in this region are small to accommodate this but Asp 265and Arg 288 are reminiscent of the enzyme’s active sites and may suggest that pertactin evolved

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from an active enzyme. However, the C-terminal part of the long loop around Leu 258 at leastpartially occludes these residues, in agreement with the absense of any obvious enzymic activity.

Pertactin has a narrow shape for its individual coils, with a short distance between PB1 and PB2and no trace of a PB1a sheet. This suggests it should be structurally aligned with either pectinmethylesterase or the P22 tailspike endorhamnosidase. However, alignment with the endorham-nosidase is difficult because the angle between PB2 and PB3 of the endorhamnosidase at T2 ismore open. Thus PB3 does not fit well when PB1 and PB2 are aligned. The parallel b-helix in theendorhamnosidase is also more curved than in pertactin. By contrast PemA aligns rather well withpertactin’s N-terminal region. Five different alignments were tried giving RMSDs in (A (withnumber of Cas aligned in brackets) of 1.93 (155), 2.04 (157), 1.79 (141), 1.99 (156) and 1.91 (154)as PemA is moved one coil at a time towards the N-terminus of pertactin. The PemA sheets areslightly more twisted than those of pertactin but it is now possible to use a single rotation andtranslation matrix to correctly align the whole parallel b-helix. The turns of this N-terminal regionof pertactin also resemble those of PemA surprisingly well. However, there is no evidence ofconserved sequences. Even when both have unusual features such as a buried polar residues whichcan be aligned at T3 (T78 and N95 of pertactin and T114 and N131 of PemA) the residues formdifferent detailed interactions.

As we move towards the C-terminus of the pertactin parallel b-helix, the sheets rotate slowly, asexpected from their twist. The shape then changes, with first the L shape disappearing as the T3region becomes smaller, the T2 turn becoming sharper and the T1 region expanding. This isrelated to the change to triangular shape in polygalacturonase but differs because of the expansionof T1. The requirement for a small residue at the a–R position at the start of PB1 is lost andresidue 340 is a leucine. In the next coil the a–R turn vanishes and the turn goes to the end of PB3near Gly 361. The coil is now almost a sandwich or a thick b-roll but PB3 persists and T1 is longenough to form almost a parallelogram. Then the T1 region is cut short but the thick sandwich ismaintained. Two tryptophans are stacked from the end of PB3 (W389 and W406) and Gly 391occurs at the turn. The coils then return to a triangular form of three sheets as PB3 and PB1lengthen, the turn at T1 changing into a very simple a–R turn. There is no sign of an indentationto give an L-shape. The stacked a–R turn of residues Asp 397, Gly 415, Asn 442 and GIn 468 maybe the most interesting feature of this region. However, another interesting feature is the turn atT3. A very similar turn can also be found in the left-handed parallel b-helix proteins and thecomparison is discussed in Section 2.8 because of its relevance to the question of how the hand ofthe b-helix is determined. Finally there is a return to the sandwich form and then the appearanceof anti-parallel b-sheet as a C-terminal b-hairpin folds back to cap the parallel b-helix.

2.5. Glutamate synthase

Binda et al. (2000) recently determined the structure of glutamate synthase from Azospirillumbrasilense at 3.0 (A resolution. This is a very large enzyme of 1472 amino acids with a four domainarchitecture. The C-terminal domain from residues 1203 to 1472 of the mature enzyme has a right-handed parallel b-helical fold with 7 coils. This domain does not seem to have a directinvolvement in catalysis or electron transport but plays a critical structural role and stabilises thedomain interface where ammonia is tunnelled between the active sites. The b-helix is reported tobe regular but not to resemble the others in the Protein Data Bank and seems to have a

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hydrophobic interior. The nature of the turns between the sheets has not yet been reported but thefigures in the article seem to suggest a slight resemblance to the insulin-like growth factor receptordescribed below.

2.6. The antifreeze protein from Tenebrio molitor

Liou et al. (2000) report that the antifreeze protein from Tenebrio molitor, TmAFP, forms anexceptionally regular right-handed parallel b-helix. This is the highest resolution parallel b-helixstructure so far solved and with two molecules in the asymmetric unit, excellent refinementstatistics and very similar structure in several coils, the unusual structure is very precisely defined.The helix is much the smallest known with 12 residues per coil and is not obviously related to anyother structure. Each coil forms a rectangle with two adjacent sides formed by a short b-sheet ofthree residues and two residues in b-sheet conformation but forming only a single conventional b-sheet hydrogen bond as shown in Fig. 9. These ‘‘sheets’’ have an aL residue between them makingan almost exactly right angle turn which is a typical T2 turn (Fig. 4a). The two remaining sides areformed of irregular structures related to b-turns, which is repetitive in the sense that it repeats ineach coil. Arbitrarily taking residues 39–50 as a representitive coil (and thus aligning with thenumbering of Liou et al. used below as 10–120), the Ramachandran (phi, psi) angles show therepeat as b–b–b–aL–b–b–aL–[�82.2, 56.9]–[�66.6, �20.4 (near 3/10 helix)]–[�125.1, 14.0]–[�66.1, 141.6 (polyproline II)]–[�84.2, �23.2 (near 3/10 helix)], where angles not obviously a or bare given in square brackets with some nearby regions indicated. Automatic assignment of thisstructure by PROMOTIF (Hutchinson and Thornton, 1996) defines a coil as:

b-sheet–type IV b-turn–g-turn–type I b-turn–type VIII b-turn

PROMOTIF does not accept the shorter b-sheet because of its unconventional hydrogen bonds,with the first residue’s NH bonding to the Og of a serine from the next coil while the carbonyl ofthe second residue bonds to the main chain NH of the residue after the next aL turn (i.e. one offset

Fig. 9. Stereo image of a single coil of the antifreeze protein from Tenebrio molitor with carbons coloured khaki,

nitrogens blue, oxygens red and sulphurs yellow. Residues 35–46 and a disulphide bridge are shown. The internal cavityis shown with the contact surface coloured by the nearest atom of the protein. The ice-binding threonines are at thebottom of the figure.

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from the expected pattern). The turn between these ‘‘sheets’’ is very similar to the T2 turn of thelyases but perhaps the best analogy is to pectin methylesterase, where Ser 249 and Thr 294 showthe same hydrogen bonding (cysteines 192 and 212 in the preceeding coils have the sameconformation but are dubious hydrogen bond acceptors while Ser 330 participates via a watermolecule in the next coil). Thus there is an analogy between these regions and PB2 and PB3 of thepectinases. The definition in terms of the b-turns is almost equally dubious because there are no b-hairpins. The unusual nature of the antifreeze protein’s main chain is shown by the startling G-value for a good 1.4 (A resolution structure of �0.46 for the Phi–Psi distribution given byPROCHECK (Laskowski et al., 1993)!

The interior of the parallel b-helix has no space for large hydrophobic residues. Stability isensured by the formation of the disulphide bridge between Cys 20 and Cys 80 and although severaloxygens and nitrogens are buried, most form hydrogen bonds. The coordinates deposited do notinclude any water molecules but Liou et al. (2000) describe the presence of an internal boundwater with low B factors between the six most regular coils occupying the cavity shown in Fig. 9.This enables the carbonyls of Cys 80 and possibly Ala 110 from one coil and the NH of 100 andperhaps 110 from the next coil to form hydrogen bonds. Apart from bridges via water, thehydrogen bonds between the coils are from NH of 10 to the carbonyl of 120 (i � 13), those of the b-sheet, NH of 40 to the carbonyl of 30 (i þ 11), NH of 50 to the Ser Og of 50 (i þ 12), the carbonyl of50 to the NH of 60 (i þ 13), the carbonyl of 60 to the NH of 80 (i � 10) and NH of 120 to thecarbonyl of 120 (i � 12). In some coils there is also a hydrogen bond from a Thr Og at 100 to the 90

carbonyl (i � 13). There is only one main chain hydrogen bond within the coil from the NH of 110

to the carbonyl of 80. Thus several peptides are close packed without hydrogen bonding to eachother. Stacking within the b-helix is restricted to the cysteines and the serines. However, thethreonines of the sheet form an obvious external stack. There are two stacked aromatics, Phe 59and Tyr 71, at position 120 and there are also hydrogen bonding residues at 70 forming an irregularladder.

The function of the antifreeze protein almost certainly involves the single well defined b-sheetwith Thr 10 and Thr 30. The b-sheet is flat, that is both unbent and untwisted. This causes the twoThr Ogs and a line of bound water molecules to lie on an array that approximately matches boththe primary prism plane and (less perfectly) the basal plane of ice. Crystal packing causes ice-likewater structure to be unexpectedly clear in the crystal structure because two two-fold relatedmolecules pack with the threonines facing each other (although TmAFP is monomeric insolution). Liou et al. (2000) argue that TmAFP functions (like other antifreeze proteins) bybinding to the surface of ice crystals so as to inhibit further growth. From the occurrence of twodifferently folded antifreeze proteins amongst the parallel b-helices, Liou et al. suggest thatparallel b-helices suited to this function.

2.7. The leucine-rich repeat family

Leucine-rich repeat (LRR) structures are characterised by a rather variable motif such asLxxLxxLxLxxNxLxxLpxxoFxx (Buchanan and Gay, 1996; Kajava, 1998) but varying from20 to 30 residues in length. Porcine ribonuclease was the first of these structures to bedetermined by Kobe and Deisenhofer (1993), revealing a spectacular horseshoe of 16right-handed coils. These structures have been described in reviews by Kobe and Deisenhofer

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(1995a, b), Kobe (1996) and Kobe et al. (1999) and thus our description will focus on therelationships and differences between LRRs and the classical parallel b-helices. We willnot discuss the LRR variant structure solved by Peters et al. (1996), which is a member of thecoiled helical folds and is not closely related to the parallel b-helices. We also usethe nomenclature of PB1, PB2 and PB3 below (sometimes with quotes when the correspon-dence is very poor) to illustrate the relationships between LRR and parallel b-helicalstructures. However, this nomenclature may not be the best description if the LRR structuresare treated separately.

2.7.1. Ribonuclease inhibitorsThe structures of porcine ribonuclease, both free (Kobe and Deisenhofer, 1993) and in a

complex with ribonuclease (Kobe and Deisenhofer, 1995b), are known and the structure of thecomplex of human placental ribonuclease with angiogenin at 2.0 (A resolution has beendetermined more recently (Papageorgiou et al., 1997). As expected, these structures are broadlysimilar and the two complexes form with the ribonuclease or the homologous angiogenin insidethe horseshoe in contact with the same gross regions of the inhibitor. The two porcineribonuclease structures show small local differences in structure which lead to quite largedisplacements when they are superposed (RMSD of 1.46 (A), corresponding to a widening of thehorseshoe opening. Surprisingly, the human ribonuclease complex with angiogenin is more similarto the free porcine structure with RMSDs of 1.24 (A against free and 1.88 (A against the complex.Also the details of the intermolecular interactions are very different with only the interactionwith the highly conserved catalytic Lys 40 of ribonuclease conserved as a general point ofattachment throughout the family. However, the 77% sequence identity means that thearchitecture of these structures is very similar with the caveat that the overall shape clearly showssome flexibility.

Each coil of RI has an almost rectangular shape with a strand of the 18 strand parallel b-sheeton one edge, an a-helix on the opposite edge and two shorter connecting regions mostly in anextended conformation without forming regular b-sheets. It is possible to align coils of theclassical parallel b-helices with a coil from RI so that PB2 matches the b-sheet and isapproximately the same length. The connecting loops then match PBla and PB3 and the a-helixmatches PB1. PB3 is longer and PB1 shorter than the matching structure so that the L-shape ofthe parallel b-helix is not seen in RI. There is some detailed similarity at the turns at the ends ofPB2 where the a–L conformation occurs as well as the occurrence of both stacked and alignedresidues on the b-sheet and the residues immediately before and after the turns. The aliphaticstacks are naturally dominated by leucines which pack against two leucines from the a-helix.There are no aromatic stacks. The polar alternating stack of asparagines and cysteines formssimilar hydrogen bonds to those seen in the asparagine ladder at the equivalent position in thepectate lyases (note that cysteine is also frequently found at this position in parallel b-helixproteins).

The b-sheet of RI shows even less twist than the slightly twisted sheets seen in the right-handedparallel b-helix proteins. However, the a-helices in each coil of RI cannot pack as close as the PB1strands of a parallel b-helix and this is accommodated by bending the sheet into the horseshoe (itmight also be possible to design a structure in which the lengths of the loops alternate as in thespiral folds described in Section 2.10 and retain a flat b-sheet).

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2.7.2. The GTPase-activating protein Ma1P from Schizosaccharomyces pombeThe crystal structure of the GTPase-activating protein Ma1P from Schizosaccharomyces pombe

(Hillig et al., 1999) resembles the ribonuclease inhibitor family in its overall fold, but is shorterwith ‘‘only’’ 11 coils. As in the ribonuclease inhibitors, each coil has an a-helix as well as a b-sheetwhich forces the structure into a crescent. A superposition of map1p on human ribonucleaseinhibitor gave an RMSD of 2.4 (A for 313 Ca atoms. In general the 11 coils are very similar withonly three repeats, LRR1, LRR3 and LRR5, identified as deviating from the consensus structure.The coil LRR3 and especially Arg 74 at the centre of the most divergent region, is identified ascritical for activating the GTPase by mutagenesis. Sequence comparisons also suggests that apatch of invariant or conservatively mutated exposed residues in this region after ‘‘T2’’ are likelyto be involved in the biological function. It is not yet clear if Arg 74 is solely involved in protein–protein interactions or if it is a catalytic residue in the active GTPase complex.

2.7.3. Human insulin-like growth factor receptorGarrett et al. (1998) determined the structure of a fragment (residues 1–462) of the insulin-like

growth factor receptor at 2.6 (A resolution, which showed two closely related examples of a foldmuch more closely related than the other LRR folds to the parallel b-helices. The insulin-likegrowth factor receptor is one of a large class of growth factor receptors coupling binding of amessenger at the exterior of a cell with signal transduction to the cytoplasm, generally viaactivation of a tyrosine kinase. The fragment consists of three domains: an N-terminal parallel b-helix domain (L1), a central cysteine-rich region and a C-terminal parallel b-helix (L2) andcorresponds to approximately half of the extra-cellular region of the receptor but does not bindinsulin-like growth factor. However, mutation within the equivalent L1 domain of the insulinreceptor does reduce insulin binding (Rouard et al., 1999). The two domains L1 and L2 are clearlyrelated and can be superimposed with RMSD of 1.6 (A for 109Ca.

These domains contain five complete right-handed coils plus an extra extended strand and forma bridge between the LRR structures such as the ribonuclease inhibitors and the parallel b-helixproteins. It is possible to align the central five strand b-sheet of L1 or L2 with PB2 and this timethe connecting regions also form hydrogen bonds to suggest a real b-sheet which aligns with PB1aand a few hydrogen bonds corresponding to PB3. The central sheet is flat because only some ofthe strands matching PB1 form a-helices. However, even when these strands are mostly extended,they do not form the hydrogen bonds of a b-sheet. The shape of each coil is closer to RI but thebending of the RI sheets causes superposition of RI to be as poor as a superposition on a parallelb-helix (polygalacturonase) (with RMSD of 2.5 (A for 60 and 62Cas; respectively). Compared withother LRR proteins, the stacking of residues is more like that seen in the parallel b-helices asisoleucine and valine often replace leucine (for example residues Ile 9, Ile 31, Leu 57, Ile 89, Ile 113and Ile 139 in the interior and Ile 2, Val 24, Val 50 and Val 75 outside but buried by interactionwith the cysteine-rich domain). The Cys–Asn alternating stack has been replaced by an asparagineladder at T2, resembling that of the pectate Lyases (Fig. 5a). The turn between PB1a and PB2often uses glycines such as Gly 27 and Gly 109 in L1 and Gly 328, Gly 356 and Gly 414 in L2. Theturn from ‘‘PB1’’ to ‘‘PB1a’’ is a rare example of an a–R turn formed by residues 23, 49, 74, 104 inL1 and residues 324, 352 and 376 in L2. However, only ‘‘PB1a’’ is a b-sheet. This turn may bestabilised by one of the two disulphides in each domain between Cys 3 and Cys 22 (L1) and Cys302 and Cys 323 (L2) at the N-terminal end of the domains.

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Possibly because the L1 domain interacts closely with the cysteine-rich domain, the L2 domainis in general more regular than the L1 with a longer asparagine ladder and a more regular C-terminal a-helix. However, L1 has the only (external) pairs of stacked aromatics (Tyr 28 and Tyr54 and in a more edge to face conformation Phe 58 and Phe 90). The face loosely equivalent toPB1 is a mixture of a-helical and irregular in both L1 and L2.

However, L1 has less helix but includes some repeating units of main chain such as the three b-turn-like conformations centred on residues 46–47, 71–72 and 101–102 which seem to extend ouridea of what can be efficiently stacked. In particular Pro 46 and Pro 71 show one way that prolinecan be included in a repeating structure.

2.7.4. Human spliceosomal protein U2A0, Rab geranylgeranyltransferase and the mRNA exportfactor TAP

The structure of the human spliceosomal protein U2A0 was determined by Price et al. (1998) aspart of a complex with U2B00 and RNA. At first sight, this structure is generally similar to theLRR region of the insulin-like growth factor receptor in that the ‘‘PB2’’ sheet is not stronglytwisted and only slightly bent. One alignment of U2A0 with the more regular C-terminal region(domain 3) gave an RMSD of 2.1 (A for 78Cas; which is clearly closer than the alignment of eitherwith a parallel b-helix structure or with ribonuclease inhibitor. However, this is essentially analignment of the b-sheet alone because the irregular or helical region of one molecule does notsuperpose on the equivalent region of the other molecule. Instead, this pair of structures shows anentirely new evolutionary possibility seen nowhere else amongst the coiled folds, in which the tworegions of the structure have slipped by one coil relative to one another. Thus the b-sheet residuesA68–A73 of U2A0 aligns on A357–A362 of IGR but residues A365–A370 of IGR superpose ontoresidues A53–A58 of U2A0. Similarly, residues A76–A80 of U2A0 superpose onto A398–A402 ofIGR as shown in Fig. 10. Naturally superpositions of a complete coil can be forced and these doseem to roughly align several leucines. However, fewer residues are aligned with a higher RMSD(i.e. 44Cas with an RMSD of 2.2 (A) and the planes of the b-sheets do not coincide. In general, as

Fig. 10. Comparison of the folding of the insulin-like growth factor receptor domain 3 and human spliceosomal proteinU2A0. Only backbone atoms are shown, excluding carbonyl oxygens. The complete U2A0 chain is shown as thin bonds

with residues 1 and 163 marked N and C, respectively. The residues 298 and 458 of the insulin-like growth factorreceptor defining domain 3 are marked and the domain is shown with thick bonds. The molecules were aligned by fittingthe backbone atoms of residues 22–27, 46–50, 68–73, 92–98 and 117–122 of U2A0 against residues 307–312, 329–333,357–362, 389–395 and 415–420 of IGR. These residues are all part of the main b-sheet.

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might be expected from the structural differences there are very few identical residues aligned byany of the possible alignments. One apparently common feature is that both have an asparagineladder at the standard ‘‘T2’’ position. However, the detailed pattern of hydrogen bonds in thisregion are very different as shown in Figs. 5a and b. This shows how a residue in each coil of U2A0

forms hydrogen bonds to the asparagine –NH2 group, rather than participating in PB2, and theasparagine side chains hydrogen bond only to main chain atoms rather than to each other.

Rab geranylgeranyltransferase, RabGGT, comprises three domains: a helical domain, animmunoglobulin domain and a C-terminal LRR domain from residue 418 to 567 (Zhang et al.,2000). The 2.0 (A resolution structure showed that the LRR domain is clearly similar to that ofU2A0 with an RMSD of 1.2 (A over 116Ca atoms from Zhang et al. (2000) (1.38 (A over 122Caatoms using O). The similarity is especially clear at the C-terminal coil despite that having adifferent conformation to the other coils. No relationship has yet been suggested between thefunction of RabGGT and U2A0.

The structure of a fragment (residues 102–372) of the mRNA Export factor TAP wasdetermined by Liker et al. (2000) in two crystal forms at 2.9 and 3.15 (A resolution. There are twoglobular domains in this fragment. Residues 119–198 have a ribonucleoprotein (RNP) fold andresidues 203–362 have an LRR fold and show homology to U2A0 (the RNP domain ishomologous to U2B00). The homology extends beyond the LRR region and includes the C-terminal extension and the Tyr 131–Asp 146 interaction of U2A0. Thus the structure is verysimilar to that of U2A0 with an RMSD of 1.56 (A for 103Ca atoms.

Like U2A0, which does not bind RNA in vitro, the isolated LRR domain of TAP did not bindRNA. Thus in both systems the role of the LRR domain is to bind the RNP domain and, whilethe RNP domain of TAP does bind RNA, in both systems association is essential for the specificco-operative binding. Mutation of the surface equivalent to that involved in the U2A0 to U2B00

interaction, suggested that the interaction survived the introduction of several alanines but wasdisrupted by reverse charge mutants. However, surprisingly some mutations at the concave b-faceof the LRR domain did not prevent the RNA interaction. The C-terminal region of the LRRdomain seems to be the critical region for the RNP-LRR binding.

2.7.5. Internalin B from Listeria monocytogenesThe recent 1.86 (A structure of internalin B (InlB) from Listeria monocytogenes (Marino et al.,

1999) is the highest resolution structure of an LRR protein currently available (from crystalsdiffracting significantly beyond the nominal resolution). Due to its resolution, this structureperhaps best reveals the structural principles of the LRR family. The central feature is the packingof the Leu–X–Leu sequence at the centre of the main b-sheet (‘‘PB2’’) with w1 angles ofapproximately 1801 and �601, respectively. These leucine pairs then stack onto the equivalentresidues in the neighbouring coils. The other leucines of the motif then pack rather less regularlyonto the leucine pair. Only the insulin-like growth factor receptor L1 and L3 domains havediverged away from this very regular packing so that this pair of leucines are no longer conserved.

Structural alignment reveals that InlB is very similar in its architecture to U2A0 (especially at itsN-terminal end) but is rather more bent. Thus the first five coils and the extra loop and b-sheet ofU2A0 superpose on the N-terminal coils of InlB with an RMSD of 1.92 (A for 102 residues andmany identical residues (especially leucines and asparagines) are superposed. The asparagineladders of InlB and U2A0 are similar as is the general form of each coil. However, InlB is longer

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and has 7 complete coils and an additional b-strand and loop region. Thus it has two extra coils,which can be assumed to be at the C-terminal end as this region is more bent and has largerhelices. The LRR repeat of InlB is 22 residues rather than 24 in U2A0 and InlB has no a-helicesbut has short stretches of 310, helix in each coil running anti-parallel to the main b-sheet. Anaspartate at position 14 of the repeat (counting the asparagine ladder as 10) may help stabilise thishelix. The resolution is sufficient to define a regular water structure of spines of waters withrelatively low temperature factors which bridge the extended regions of the structure. Thus in thenon-helical regions anti-parallel to ‘‘PB2’’, the main chains do not directly form hydrogen bondsand are significantly further apart. The result of this and the 310 helices is that the LRR region isbent by approximately 141 per coil.

InlB has a calcium binding N-terminal cap of residues 36–76 but no C-terminal cap. Thehydrophobic core of the cap is continuous with that of the LRR domain. The calciums do notappear to be necessary for the folding of the structure and are rather weakly bound. However,they may form bridges in the interaction of InlB with its ‘‘receptor’ or target.

Marino et al. (1999) proposed that the concave faces of the LRR structures make themespecially suitable to make protein–protein interactions. Marino et al. (2000) aligns the internalinsand begins the assignment of possible functional residues and binding sites.

2.8. Left-handed parallel b-helix structures

2.8.1. Left-handed parallel b-helix structures containing hexapeptide repeatsFive left-handed parallel b-helix structures containing hexapeptide repeats have been

determined: UDP-N-acetylglucosamine acyltransferase, LpxA, from E. coli (Raetz and Roderick,1995), a carbonic anhydrase, Cam, from Methanosarcina thermophila (Kisker et al., 1996),tetrahydrodipicolinate N-succinyltransferase, DapD, (Beaman et al., 1997, 1998a), a xenobioticacetyltransferase, PaXAT, from Pseudomonas aeruginosa (Beaman et al., 1998b) and thebifunctional N-acetylglucosamine 1-phosphate uridyltransferase from E. coli (Brown et al., 1999;Olsen and Roderick, 2001) and from Streptococcus pneumoniae (Sulzenbacher et al., 2001). Theseare all members of a large family of enzymes with a hexapeptide repeat motif [LIV]-[GAED]-X2-[STAV]-X (Vaara, 1992). Most function as acyl transferases, transferring acetate, succinate or R-3-hydroxy fatty acids using either acyl-coenzyme A or acylated acyl carrier protein. Thedifferences between this area of mammalian and bacterial metabolism have inspired great interestin finding inhibitors of these enzymes as potential antibiotics.

The left-handed parallel b-helix of these enzymes resembles a triangular prism with the flat butpleated b-sheets forming the faces. The active form of all these enzymes is the trimer and the modeof trimerization is generally conserved. Four of the five enzymes form trimers; with the axis of theparallel b-helix aligned with the three-fold axis to within a few degrees. However, PaXAT has anangle of 211 between the helix and the trimer axis. Beaman et al. (1998b) suggest that thismisalignment is possible because the parallel b-helix in PaXAT is the shortest of the knownstructures. LpxA has 10 coils, DapD and Cam have 7 coils and PaXAT only 5 coils. The form ofN-acetylglucosamine 1-phosphate uridyltransferase used for the initial structure determination(Brown et al., 1999) was truncated after residue 331 of the 456 residue enzyme because the crystalsfrom the complete molecule did not give satisfactory diffraction. This truncated molecule had thepyrophosphorylase activity of the second step of the overall synthesis of Udp–GlcNAc, which is

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associated with the N-terminal domain, but had lost its acetyl transferase activity and had only 4complete coils and 13 of the 23 hexapeptide repeats. Subsequent structures (Kostrewa et al., 2001;Olsen and Roderick, 2001; Sulzenbacher et al., 2001) revealed that N-acetylglucosamine 1-phosphate uridyltransferase has the longest known left-handed parallel b-helix This left-handedparallel b-helix has 10 complete coils without including an N-terminal helix, which plays a similarrole to that of the pectinases, and an extra b-strand. Then there is a remarkable ‘‘domain swap’’ inwhich a C-terminal extension add an extra b-strand to extend the parallel b-helix of its symmetryequivalent in the trimer. Thus we could generously argue that there are 11 complete coils.However, the regularity of the hexapeptide repeat is less in this structure because there are severalsingle residue deletions and one large insertion at the T3 turn, which is the probable site of theacetyltransferase activity. An interesting difference between the truncated and wild-type enzymesfrom E. coli is that there is a disulphide bridge inside the b-helix only in the truncated enzyme,which Olsen and Roderick (2001) suggest is due to the increased accessibility of these cysteines inthe truncated form.

The active sites of DapD and of PaXAT have been identified by binding substrates andsubstrate analogues (Beaman et al., 1998a, b) and lie at a monomer–monomer interface. Theactivity involves residues from both molecules. This is also the probable active site of LpxA(Wyckoff and Raetz, 1999) and of the acetyltransferase activity of N-acetylglucosamine 1-phosphate uridyltransferase, which Pompeo et al. (2001) have shown requires trimers for activity.Similarly the zinc at the active site of Cam has three histidine ligands from two molecules at asimilar distance from the three-fold axis. Thus we would expect to find trimer formation to beconserved in the evolution of all these enzymes. Mutants with altered activity have been made ofserine acetyltransferase (Wirtz et al., 2001) who also constructed a model from the knownstructures and thus identified the same active site. Crystals of serine acetyltransferase had beenreported (Wigley et al., 1990) but only the symmetry of the structure has so far been reported(Hindson et al., 2000).

The left-handed cross-over connections between the b-strands of these structure weresurprising. Richardson (1976) had noted that such connections are very rare and argued thatthe inherent right-handed twist of extended polypeptides and of a-helical segments naturally foldsa protein into a right-handed coil, as the ends of these segments are brought together. In the left-handed parallel b-helix this may not be important because the sheets are unusually flat and theconnections between adjacent b-strands are long. This leaves the question of why the chain foldsinto a left-handed rather a right-handed helix.

Kisker et al. (1996) suggested that the origin of the chirality was the a–L turns. At first sight thissuggestion is immediately refuted by the prevalence of such turns in the right-handed folds.However, the turns in the left-handed parallel b-helices are different from those generally found inthe right-handed folds (for example at T2) and in fact the hexapeptide repeat can be imagined asencoding a single turn. The turns are tighter and sometimes resemble classical b-bends in forminga 1–4 hydrogen bond. Good examples can be found in the region Y63–Q64–F65–A66 of LpxAand the neighbouring coils (Fig. 6a). Residues 64 and 65 (escaping the confusion of defining i;i þ 1; etc. from the hexapeptide repeat, the b-bend or the T2 turns of pectinases) are in polyproline(proline is sometimes found) and a–L conformations, respectively. The turns are stacked withreasonable hydrogen bonds between the oxygen of the residue in polyproline conformation andthe amide hydrogen of the residue in a–L conformation in the next coil. Thus the Wilmot and

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Thornton (1990) definition of this turn would be bPgL (the classical type 11 of Richardson, 1981)rather than bEgL: However, the turn does not form a b-hairpin and the strands diverge aftermaking one hydrogen bond.

The distortion away from a simple a–L conformation and formation of the 1–4 H-bond couldbe significant, either as a cause of the chirality, or as a consequence of it. In the above example, therotation of the peptide 65–66 to make the hydrogen bond rotates the side chain of residue 66 tothe side of the side chain of residue 62, a choice which defines the overall chirality. However, thisargument may be weakened by comparing these turns with those at T3 towards the C-terminus ofpertactin. Residues 117–121 of LpxA has the sequence LMINA while residues 353–357 ofpertactin has the sequence LTGGA. Despite the GG pair the main-chain and side chains occupyvery similar positions. The five Ca atoms can be fitted with RMSD of 0.74 (A. The alanines areconserved not just in several turns of both proteins so that pertactin as A333, A357, A387 andA404. Leucine also occurs at 329, 353 and 383 and these residues are stacked followed byisoleucine 401. Similarly Leu 117 and Leu 135 are stacked in similar positions to Leu 353 and Leu329 (note the inversion), respectively. Fig. 6 shows this comparison directly after superposing allthe main chain atoms of residues 116–123 of LpxA on residues 352–359 of pertactin with anRMSD of 0.78 (A. Thus it would seem that there may be a consensus sequence for this type ofturn, even in right-handed structures, but that it does not force a chirality on the protein.

Bateman et al. (1998) suggested that the most likely origin of the chirality is the side chaininteractions. The first residue of the motif is generally L, I or V although aromatic residues arefound. Three of these residues come together and intercalate their side chains, forming a chiralpacking at the centre of the parallel b-helix. When three stacks of mostly isoleucine side chains(L44, 162, 192 and L117; V50, 168, 198 and 1123; and 156, 186 and V111, extending to V129 andV147) come together at the centre of LpxA, the result is an attractive (to a crystallographer)example of symmetric close packing (Fig. 11). The closest distances are between each Cd1 and twoCg2 atoms. For example Cd1 of Ile 68 is 4.45 (A from Cg2 of Ile 62 and 4.52 (A from Cg2 of Ile 92.

Fig. 11. The packing of side chains in the left-handed parallel b-helix family. (a) Residues 83 to 99 of LpxA showing

most of a coil and the interaction of isoleucines 86, 92 and 98. Residues 93–96 form the characteristic turn of the left-handed parallel b-helices. Carbons are drawn in light grey with oxygens and nitrogens in dark grey with some residuenumbers indicted to the right of the a-carbons. (b) Two coils of LpxA with residues 54–69 added in front of 83–99 in the

same orientation and colours as in Fig. 11a (with labels removed). Both aliphatic and polar stacks are shown. The viewis slightly off the axis of the parallel b-helix.

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However, Cg2 of Ile 68 is less symmetrically placed 3.62 (A from Cd1 of Ile 86 and 4.66 (A from Cd1of Ile 56. The imperfect 3-fold symmetry continues with Ile 86 Cg2 at 4.71 (A from Ile 92 Cd1 andIle 86 Cd1 at 4.81 from Ile 98 Cg2: All the isoleucines are in rather similar conformations: Ile 68 isslightly distorted with w1 ¼ �46; w2 ¼ �150; Ile 86 has w1 ¼ �68 and w2 ¼ þ155; Ile 62 hasw1 ¼ �66 and w2 ¼ 154: 156 has w1 ¼ �59 and w2 ¼ �177; 192 has w1 ¼ �56 and w2 ¼ þ161; 198has w1 ¼ �50 and w2 ¼ þ171: Given the sequence and the positions of the main-chain it would bepossible to find this as an optimal or near optimal solution for side-chain packing. However, it ismore difficult to demonstrate that there is not a similar packing with the hand of the b-helixreversed and to assess the importance of the chiral isoleucine side chain in defining the optimalhelix chirality.

The right-handed parallel b-helices do not have the approximate 3-fold symmetry of the left-handed family and tend to be L-shaped. The interactions across the helix tend to involve side-chains from only two b-sheets which are closer to a sandwich than to an equilateral triangle. Thefirst residue of PB1 often interacts with residues from PB2 and PB3 which are themselves incontact with each other. The hand of the helix is defined by how these side chains interdigitate butthe altered main chain orientations and the lower symmetry make it hard to make a directcomparison. The triangular region of pertactin seems more similar but the presence of aromaticresidues and lower symmetry again makes comparison difficult. However, residues Leu 417–Val425–Leu 439 or Leu 444, Phe 450 and Leu 464 of pertactin could suggest that the isoleucines ofthe left-handed family are critical. Isoleucines are found at the first position of the hexapeptiderepeat in all these left-handed structures but the packing is generally less symmetric than that ofLpxA.

It is interesting that the right-handed parallel b-helix proteins except pertactin have a-helices attheir N-termini while the left-handed parallel b-helix proteins have a-helices at their C-termini.However, the N-acetylglucosamine 1-phosphate uridyltransferase shows that these helices are notessential for establishing the overall chirality and also that more than half the helix can beremoved without changing the folding of the N-terminal region.

Stacking is even more obvious in the left-handed family than in the right-handed because of therepetitive sequence but is mostly restricted to aliphatic residues. Polar residues are rare in the coreof left-handed parallel b-helices and there are no asparagine ladders. Aromatic residues are foundoccasionally at position i; although there is not enough room for three large residues at position i:Only a single two residue internal aromatic stack has been observed in PaXAT (Phe 71 and Phe125). In the right-handed parallel b-helices aromatic stacks may be favoured by the twist of thesheets as the rings must stack with sufficient offset so that the electron-rich centres do not repeleach other too strongly. However, there are external aromatic stacks in the left-handed parallel b-helices, suggesting that either space limitations are critical or that aromatics do not favour the left-handed fold.

2.8.2. The left-handed parallel b-helix antifreeze protein from spruce budwormThe structure of the spruce budworm antifreeze protein, SbwAFP, has been determined by

Graether et al. (2000) and is the first parallel b-helical protein structure determined by NMR. Ithas a smaller coil than the hexapeptide repeat proteins of Section 2.8.1 and can be considered as a‘‘pentapeptide repeat protein’’ with the caveat that the repeats are less regular. SbwAFP has 90residues and the parallel b-helical region only extends to residue 72 and is followed by some anti-

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parallel strands. There are only four coils making this the least repetitive parallel b-helix so farobserved. It is also one of the least regular parallel b-helices seen although stacking probably doesoccur inside the b-helix with examples such as Val 32, Ile 47 and Ile 64. It is unusual in having avariety of types of turn. It is triangular and the spacing between the b-sheets (one of which ispoorly defined as described below) is very similar to that seen in the other left-handed parallel b-helices. Because of this it is possible to make a structural alignment of SbwAFP with one of thehexapeptide repeat family. Clearly many alignments are possible but the simplest is to align thefirst b-sheet of each protein, so that residues 7–72 of 1EWW (model 1) are aligned on 4–90 of1LXA. Using O, this gives an RMSD of 1.85 (A for 68 a-carbons. However, this is essentially onlyan alignment of the b-sheets and the turns are generally very different with one less residue perturn in SbwAFP. Thus the SbwAFP again argues that the turns do not enforce the chirality of theparallel b-helix. There are isoleucines packing in the hydrophobic core of SbwAFP but there arefewer than in the hexapeptide repeat family and they are less regularly packed with aromaticresidues and disulphides inside the parallel b-helix. In the alignment above, Ile 62 and Ile 68 of1LXA are aligned with Ile 57 and Ile 64 of SbwAFP although Ile 56 of 1LXA is aligned with Ser52 of SbwAFP. Formally, Ile 68 of SbwAFP then aligns with Ile 86 of 1LXA but theconformations are quite different. However, there is another possibility that the chirality of thisfold is defined by the three disulphide bridges, Cys 25–Cys 37, Cys 62–Cys 85 and Cys 67–Cys 80of SbwAFP.

Like other antifreeze proteins, SbwAFP probably functions by binding ice nucleii to preventtheir growth. This probably involves an array of threonine residues (5, 7, 21, 23, 36, 38, 51, 53 and70) on one face of SbwAFP. In a regular parallel b-helix, many of these could form stacks. Thisparticular region of SbwAFP shows exchange broadening and thus the atomic positions on thisface are poorly defined. However, some 20–30% of the ensemble has some b-structure for thisregion. It is possible that this region becomes a regular b-sheet and presents an array of orderedstacks of threonines when the protein binds to ice. Certainly site directed mutagenesis shows thatT7L, T21L, T38L and T51L each show a 80–90% loss of activity.

2.9. Parallel b-rolls

The alkaline protease of Pseudomonas aeruginosa comprises an N-terminal zinc metalloproteasedomain and a C-terminal domain consisting of a 21-strand b-sandwich (Baumann et al., 1993).Within the C-terminal domain the successive b-strands are wound into a right-handed superhelixwith calcium ions bound within the turns between a tandem repeated GGXGXDXLX. The twolayer b-sandwich architecture built by a succession of these sequence motifs is called the ‘parallel broll’ motif. The tightly bound internal calcium ions appear to lock the structure together. Thisdomain has no clear functional role although an involvement in secretion, export and receptorbinding has been suggested.

The Serratia marcescens mettalloprotease was shown to have a similar b-roll domain made oftandem repeats with the four turns stabilised by five calcium ions (Baumann, 1994). The roll isstabilised by three kinds of interactions: Firstly, by main-chain hydrogen bonds of the carbonyloxygen of residue i to the amide nitrogen of i þ 1; and the amide nitrogen of residue i and thecarbonyl oxygen atom of residue i þ 17; secondly, by hydrophobic interactions in the centre of thesandwich, mediated mostly by interdigitating leucine residues at position 8 of the consensus

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sequence (see above) and thirdly by electrostatic interactions between calcium ions and asparticacid residues at position 6 of the consensus sequence. Since the b-roll motif is made up of arepeating nine-residue sequence motif, there is an almost exact repeat in the three-dimensionalstructure after 18 residues with the exception of the edges of the b-helix (Baumann et al., 1993;Miyatake et al., 1995; Hamada et al., 1996). In this respect the b-roll differs from the parallel b-helix which has three parallel b-sheets and no calcium-ions bound within the domain architecture.

2.10. Spiral folds

The spiral fold is a family of repetitive structures with coiled or solenoid folds in which each coilconsists of two b-sheets and an a-helix and the overall sense of the fold is right-handed. The threeknown structures are two coenzyme A dependent enzymes, 4-chlorobenzoyl coenzyme Adehalogenase (Benning et al., 1996) and enoyl-coenzyme A hydratase (Engel et al., 1996), andCIpP, an E. coli ATP-dependent protease (Wang et al., 1997). In these enzymes, the two parallelb-sheets, A and B, approach each other at a similar angle to PB2 and PB3 of the parallel b-helixproteins. The problem of packing the a-helices is not resolved by bending the b-sheets as in theribonuclease inhibitors but by altering the coils so that neighbouring a-helices are at differentdistances from the b-sheets. This is one factor making these structures less regular than theparallel b-helix proteins. However, aliphatic stacks do occur such as Ile 16–Ile 53 and Val 51–Val103 of enoyl-coenzyme A hydratase or Ile 59–Val 87 of CIpP. CIpP also has an external aromaticstack of Phe 30–Tyr 62. A second factor in making the spiral folds appear less regular is that thetwo b-sheets cannot be joined by the short stacked a–L turns of the parallel b-helix proteins norby the a–R turn seen in some coils of pertactin. This is because the sheets are not ‘‘in phase’’ toallow these turns so that if sheet A is aligned with an example of PB2, sheet B forms its hydrogenbonds in the opposite direction to those of PB3. Viewed from the perspective of the parallel b-helix proteins, the spiral folds importance is to illustrate that it is possible to fold a structure whichcannot make the a–L or a–R turns. This is another example of a fold arising by repeating a simpletheme and the limited stacking may reflect this evolution.

3. The prediction and design of parallel b-helix structures

General techniques for the identification of folds from amino acid sequence (Sippl andFlockner, 1996; Jones et al., 1999) have been developed during the 1990s. The repetitive foldsseem to be a special case and it has been suggested by Yoder and Jurnak (Yoder and Jurnak,1995) that this fold might be unusually simple to identify. Heffron et al. (1998) describe a methodbased on searching with a sequence profile for a single coil of a parallel b-helix derived fromhomologues of the pectate lyase PeIC. This method fails to find the known parallel b-helixproteins, except for the pectate lyase family, because it demands that parallel b-helix proteins havethe asparagine ladder found in the pectate lyases at T2. It does find a number of protein familiesbut only the three-dimensional structure of internalin B has so far been determined (Marino et al.,1999). Internalin A and B were the proteins most convincingly identified because these were foundwith the most restrictive profile and the profile identified multiple hits as expected if these proteinshad several coils. Thus it is clear that this method does identify some repetitive structures but does

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not distinguish the parallel b-helix fold from the LRR fold. However, before 1998 only long LRRstructures were known, so it was hard to fit short repeats to this fold. It may be possible in futureto identify the LRR folds by ‘‘threading’’ sequences onto the families of LRR folds now known.

Bateman et al. (1998) have proposed that a class of proteins containing the pentapeptide repeatA(D/N)LXX fold into a right-handed parallel b-helix, with each repeat forming having theconformation bbbba–L and three sheets forming a coil. This would form the small parallel b-helixshown in Fig. 12. They note that a Fourier analysis of the sequences only reveals the pentapeptiderepeat, with no evidence for a 15 residue periodicity. However, Fourier analysis of the left-handedb-helical hexapeptide repeat proteins does not reveal any periodicity higher than 6. As the lessconserved residues 4 and 5 were likeliest to point outwards, there were two choices for the turn.Because residue 3 was assumed to point inwards, either residue 5 could be modelled as a–L, orresidue 4 as a–R. The a–L turn was preferred to avoid the side chain of residue 4 colliding with themain chain of the next coil and preventing good hydrogen bonding of the 4–5 peptide. The choiceof a righthanded parallel b-helix was made to optimise the packing of the leucine side chains(position 3) in the centre of the parallel b-helix. The recent structure of a left-handed parallel b-helical antifreeze protein (Graether et al., 2000) made up of repeating pentapeptides will naturallyinspire speculation on the relationship between these structure.

Marino-Buslje et al. (1999) have reported their construction of a model of the insulin receptor, aLRR protein, based on pectin lyase A and the phage P22 tailspike protein and compared it withthe X-ray structure of IGR (Garrett et al., 1998). In this case it was possible to identify the firstdomain of the receptor as a parallel b-helix and to deduce that it had at least 4 and probably 5coils, despite the differences between the receptor and the superfamily containing PnIA and TSP.Again the problem of identifying a short LRR fold before 1998 is apparent.

An interesting link between the pectinases and the LRR proteins is supplied by the observationthat plant polygalacturonase inhibitor proteins, PGlPs, are LRR proteins. Leckie et al. (1999)have predicted a model of the structure of PGIP from Phaseolus vulgaris based on that ofribonuclease inhibitor and used it to interpret the effects of mutation. As the same team have alsogrown crystals (Leech et al., 2000), it should soon be possible to compare this prediction with anexperimental structure.

Fig. 12. Stereo view of residues 16–46 (coils 2 and 3) of the right-handed parallel b-helix model proposed by Bateman

et al. (1998) for the pentapeptide repeat family. Residues of the second coil are numbered. Carbons are drawn in lightgrey and polar nitrogens and oxygens in dark grey.

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Collinson et al. (1999) have constructed models of AgfA, the insoluble fimbrial subunit ofSalmonella thin aggregative fimbriae and their preferred model is derived from the b-roll. Thesequence can be predicted to be mostly b-sheet and shows five clear repeats of 22–23 amino acidsexcept for the last coil with 18 residues. The model has internal stacks of both glutamine andasparagine with their amides hydrogen bonding to the peptide backbone and the asparagine’sconformation is rather similar to the asparagine ladder of the pectate lyases. However, the moreobvious similarity is to the asparagines sometimes found at the ends of the stacks of calciumbinding asparates in the b-roll structures (for example, Asn 347 of 1KAP). The model of AgfAdiffers from the b-rolls in that the tight turn and very short distance between the b-sheets is onlyseen at one end of the sheets with the asparagines and glutamines. The sheets are further apart andthe packing less interdigitated are the other end.

Lilie et al. (2000) briefly review the occurrence and possible function of the motifGGXGXDX(LF/I)X in the RTX family of toxins. This motif occurs in all the proteins secretedby the haemolysin pathway, including the alkaline protease of Pseudomonas aeruginosa where itwas shown to form the b-roll structure. They then report the design and properties of a 75-merwith 8 identical repeats of the motif, NH2–WLS–[GGSGNDNLS]8–COOH. This sequence is asoluble monomer in solution but the circular dichroism does not change on the addition of 6 Mguanidinium hydrochloride. Thus there is probably no defined structure present even whencalcium is added. Only on adding 100mM calcium chloride and 25% (w/v) polyethylene glycol8000 was a change to the spectrum expected for b-sheet observed and the peptide polymerised sothat no monomers or dimers were present. The circular dichroism spectrum and the very tightbinding of calcium suggest that the designed b-roll structure had been formed.

Liou et al. (1999) were very successful in predicting that 12 residue repeat sequences from theantifreeze proteins of Tenebrio molitor were b-helical and that the conserved threonines formed anice-binding array on a parallel b-sheet. As described above, this prediction has been subsequentlyverified. This may have been an unusually helpful case because the sequences differed by 12residue insertions and deletions but shows that at least some b-helical structures can be predictedfrom their sequences.

Graether and Jia (2001) have used the observed structure of the spruce budworm antifreezeprotein, SbwAFP, as a template to construct a model of an ice-nucleation protein fromPseudomonas syringae, INP. The basic idea is that a short parallel b-helical protein can bind to theprism face of ice via threonines and act as an antifreeze protein while a longer parallel b-helix canform a nucleus and promote freezing. However, the sequence repeat of 16 in INP rather than 18residues of the template forces the triangular structure to become more oval and the left-handedchirality is enforced only by memory of the template.

The above fold identifications from the sequences have involved repetitive sequence motifs.Initially it was much harder to identify the apparently nonrepetitive sequences of the enzymesdegrading polysaccharides. The obvious approach is classical sequence alignment assisted byknowledge of conserved residues. Pissavin et al. (1998) align PelZ with PelC from Erwiniachrysanthemi. The alignment gives 27% (96/353) identities with many gaps and is not obviouslysignificant as a single alignment. However, the overall significance is shown because the b-strandstend to be conserved and most of the critical active site residues are also present in PelZ.

In a combination of direct sequence alignment with the identification of repetition, Finnie et al.(1998) have identified a repeating motif N–(I/V)–X–(I/V)–(X)–(D/E)–N in two polysaccharide

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glycanases, PlyA and PlyB from Rhizobium leguminosarum, and in SpsR from Sphingomonasstrain 888 (Yamazaki et al., 1996). PlyA and PlyB can be aligned with the extra-cellular pectatelyase superfamily with approximately 20% identity, the sequences suggesting a high b-sheetcontent with a predicted a-helix near the N-terminus. Finnie et al. proposed that PlyA and PlyBare distant homologues of the pectate lyases. The motif occurs 10 times in PlyA and PlyB andassuming that it forms a single b-sheet, suggested that these proteins have rather longer parallel b-helix than the pectate lyases. It also seems likely that the structure needs some way ofaccommodating the D/E residue in a stack. While the prediction of a parallel b-helix fold isplausible, these enzymes have different specificities from the pectate lyases and the alignment givenonly conserves one of the conserved active site residues of the pectate lyases.

Very recently, the results of the ‘‘critical assessment of fully automated structure prediction’’ orCAFASP have become available at and the results using bothpectin methylesterase and PeIl- suggest that advances in algorithms and the availability of severalb-helical structures have almost solved the problem of recognising these folds. However, while b-helical structures are generally found as the most likely ‘‘guesses’’ for these structures, there is stillscope for progress in increasing the confidence level and improving the final alignments. It will beextremely interesting to compare the models generated with human intervention in the parallelCASP4 contest when these become available.

An alternate way of identifying the parallel b-helix fold was suggested by Sieber et al. (1995)who argued that the circular dichroism, CD, from the parallel b-helix proteins is hard to representas a sum of the normal basis sets. CD recognises secondary rather than tertiary structure and thusgenerally cannot distinguish parallel and anti-parallel b-structures (Johnson, 1999), presumablybecause there is little overlap of orbitals except along the main chain. However, effects due to thelength of secondary structure elements and their end conformations have been reported (Pancoskaet al., 1999). Thus a plausible origin for a unique parallel b-helix CD spectrum is the frequentoccurrence of a–L residues at the ends of the short b-sheets. This suggests that if there is a uniqueparallel b-helix spectrum, both left- and right-handed folds may have similar spectra if bothcontain a–L bounded b-sheets, with the caveat that the turns are rather different as shown inFigs. 4 and 6. Kamen et al. (2000) suggest an alternative explanation that the repetitiveorientation of the aromatic residues generates an unusual spectrum, although they also report thatthe conventional interpretation of the CD spectrum gives a reasonable fit to their data.

Khurana et al. (2001) made the interesting observation that the pectate lyase PelC and the P22tailspike endorhamnosidase bound Congo red and induced different bands but both with positiveellipticity while the left-handed LpxA bound Congo red and induced two bands with negativeellipticity. This raises the possibility that the chirality of the Congo red binding site can be directlyobserved. However, Congo red binds to many proteins and may not always bind to b-helices.Congo red did not bind to crystals of Erwinia polygalacturonase (RWP unpublished).

A very interesting article by Khurana and Fink (2000) discussed the Fourier transform infrared(FTIR) spectrum of parallel b-helix proteins as well as reviewing many aspects of this architectureand its possible relationship to amyloid fibrils. They concluded that FTIR could not reliablydistinguish parallel b-helix proteins from other proteins rich in b-sheet. However, they did notethat the percentage of b-sheet determined by FTIR was consistently higher than that calculatedfrom the crystal structures. They attribute this to the extra hydrogen bonds found both at turnsand in the asparagine ladders.

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4. Are amyloid fibrils related to parallel b-helices?

The formation of amyloid fibrils is a feature, and possibly an important link in the causation, ofa range of human diseases including Alzheimer’s disease., where the role of fibrils and protofibrilsin pathogenesis has been recently discussed by Selkoe (1999) and Lansbury (1999). Deposition ofamyloid fibrils of many different proteins occurs in various diseases (amyloidoses) but the fibrilsall appear to be 50–130 (A wide, rigid and either non-branching or showing infrequent branches.All give a characteristic green birefringence with the dye Congo Red under cross-polarised light.Fibrils grow from a wide range of peptides, which normally either have a biological function orarise by degradation of a larger functional molecule, or else from covalently intact proteins. InAlzheimer’s disease the fibrils are formed from a peptide fragment, typically 40–42 amino acidslong, of a much larger membrane bound protein. Fibril formation depends on the concentrationof the peptide both in vivo and in vitro. Kinetic studies suggest a nucleation-growth mechanism(Jarrett and Lansbury, 1993) and a clear distinction between nucleation and further deposition(Esler et al., 1996a, b). The monomer is reported to be the species involved in adding to theplaques (Tseng et al., 1999; Esler et al., 2000) and adds reversible followed by irreversible binding.Interestingly, the kinetic analysis of Esler et al. (2000) suggests that nucleation should require ‘‘atleast thousands of years to occur spontaneously’’. Islet amyloid polypeptide or IAPP, a 37 aminoacid peptide which is normally secreted together with insulin and whose deposition occurs in non-insulin-dependent diabetes mellitus, also shows similar nucleationgrowth kinetics of fibrilformation (Kayed et al., 1999).

Recently, a number of proteins not normally associated with any disease have been shown toform fibrils resembling classical amyloid fibrils. For example, Guijarro et al. (1998), Chiti et al.(1999), Alexandrescu and Rathgeb-Szabo (1999) and Gross et al. (1999) describe the formation offibrils from an SH3 domain of the p85a subunit of phosphatidylinositol 3-kinase, acylphospho-sphatase and the cold shock proteins CspA and CspB (see also Section 2.2 for the case of thephage P22 tailspike endorhamnosidase). This suggests that there is an ‘‘amyloid state’’ accessibleto many proteins, especially in the presence of low concentrations of denaturants. Proteins haveapparently evolved to fold rapidly under ‘‘natural’’ conditions without generating significantconcentrations of partially folded intermediates and there may often also be a significant energybarrier to the formation of the amyloid state (Kusumoto et al., 1998) at least under nativeconditions. The mutations known to be associated with hereditary amyloidoses seem to act bydestabilising the native state (Kelly, 1998; Dobson, 1999; Radford and Dobson, 1999) and thusfavouring partially folded conformations. The refolding of recombinant proteins from inclusionbodies offers an obvious parallel. Another approach to the requirements for amyloid formation isvia combinatorial peptide libraries (West et al., 1999), which shows that many sequences with analternating pattern of hydrophobic and hydrophilic amino acids can form fibrils. West et al. alsofound in a survey of heptapeptide sequences in native proteins with four hydrophilic and threehydrophobic residues that the alternating pattern occurred least frequently of the 35 possibilities.Kalberg et al. (2001) argue that an important predictor of a protein’s ability to convert to amyloidis that the folded structure contains an a-helix which could be predicted from the sequence as b-sheet. However, it may be important to notice that not all conversions to b-sheet lead to amyloidfibrils. Takahashi et al. (2000) show that quite similar peptides may produce or not produceamyloid on conversion and that the kinetics of conversion are very sensitive to mutation.

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The classic method for the analysis of the structure of amyloid fibrils has been X-ray fibrediffraction. Models have been proposed by Blake and Serpell (1996) and Blake et al. (1996) fromanalysis of fibrils from the Val30Met mutant of human transthyretin, which gave relatively highresolution. The diffraction from transthyretin is compared with that from amyloid from severalsources by Sunde et al. (1997) and in a more general review by Sunde and Blake (1997). Theyconcluded that all the fibrils are formed from protofilaments of 25–60 (A in diameter. The fibrilsare predominantly composed of b-structure although the three-dimensional structures of thenative proteins are sometimes predominantly a-helical. The X-ray diffraction pattern is thecharacteristic cross b-structure, implying that the b-sheets lie approximately perpendicular tothe fibril axis. The diffraction pattern and thus the model does not constrain whether the b-strandsare parallel, anti-parallel or mixed. The degree of twist between adjacent strands is also not directlyaccessible but Blake and Serpell’s model uses the average twist seen in globular protein structures(about 151) to account for a 115 (A repeat distance seen along the fibre axis as a 24 strand repeat.

Malinchik et al. (1998) have analysed fibre diffraction and fibril formation from Alzheimerb-peptides (Ab below) and compared Ab (1–40) with shorter peptides such as Ab (1–28), Ab (11–28), Ab (13–28), Ab (11–25), Ab (9–28), Ab (19–28), Ab (34–42), Ab (22–35) and Ab (25–35).Their indexing of the diffraction pattern is different but their model is generally similar to Blakeand Serpell’s model and the conclusion that fibrils are formed from 3–5 protofilaments, in thiscase approximately 30 (A in diameter, is also similar. However, Malinchik et al. conclude that thehydrogen bonds of the b-sheet are not exactly perpendicular to the fibril’s axis. Distinct twistedand flat fibrils were identified by electron microscopy using negative stain but both gave similardiffraction spacings.

Cryo-electron microscopy was used by Jimenez et al. (1999) to examine the fibrils from the SH3domain at 25 (A resolution. These fibrils are twisted with two pairs of protofilaments wound as adouble helix around a hollow core. The helical crossover repeat was B600 (A and there was anaxial repeat of B27 (A. The protofilaments were 20 (A by 40 (A and thus flatter than the native SH3domain. Thus even an all b-protein has extensively refolded in forming the fibril. It is argued thatthe protofilaments are so flat that they cannot be included more than a b-sandwich of twoessentially flat sheets (> 21 twist between strands).

The identification of protofilaments as components of fibrils can be contrasted with theidentification by Walsh et al. (1997, 1999) and Harper et al. (1997, 1999) of independently existingprotofibrils of the Ab peptide, which are probably intermediates in fibril formation. Protofibrilsgrow from Ab (1–40) after aggregates have been removed by either size exclusion chromatographyor dissolution in DMSO and filtration. The dimensions of these flexible rods were determined byatomic force microscopy, AFM (Harper et al., 1999) or electron microscopy and quasi-elasticlight scattering (Walsh et al., 1997) with reasonable agreement. Both groups suggested that it maybe the protofibrils which are critical for Alzheimer’s Disease pathology. AFM has also been usedto characterise the formation of fibrils of immunoglobin light-chain amyloid by coiling of two orthree protofibrils (Ionescu-Zanetti et al., 1999), suggesting that protofibrils are intermediates inthe formation of most amyloid fibrils. The review of Rochet and Lansbury (2000) gives anexcellent account of this rapidly developing research.

Fourier transform infrared spectroscopy (FTIR), especially when combined with isotopiclabeling (Anderson et al., 1996), can distinguish parallel and antiparallel b-structure and givedetailed information on the orientation of amide carbonyls. Lansbury et al. (1995) combined

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FTIR with solid state NMR to analyze the fibrils formed from the C-terminal Ab (34–42) peptide,using 13C labelling. The 13C NMR line widths suggest that the environment of the peptide in thefibril is less uniform than a crystalline solid, but more uniform than a typical amorphous material.As the conformations do not exchange on the NMR time scale, most of the peptide may beinaccessible to water. By diluting the 13C labelled peptide by unlabelled peptide, it was possible toobtain intramolecular 13C–13C distances and these were used to generate models of the monomer.These were all highly pleated extended conformations and hairpin folds could be excluded.Lansbury et al. conclude that the b-sheets are intermolecular and that the intermolecular distancesshow that two different interactions occur. In a subsequent study of the same peptide Costa et al.(1997) eliminated the possibility that a cis peptide occurs between residues Gly 37–Gly 38, leavingmodels with two regions of highly pleated anti-parallel b-structure on either side of an unknownconformation for the glycines. The central residue 20–29 peptide of the islet amyloid polypeptide(IAPP) has been shown to form highly pleated b-structure by solid state NMR (Griffiths et al.,1995). The intermolecular packing was constrained by an intermolecular rotational resonancebetween the Ca of Ala25 and the C=O of Ile26.

Benzinger et al. (1998) studied the peptide Ab (10–35)NH2 by solid state NMR. Ab (10–35)NH2 was more soluble than the complete peptide Ab (1–42) but is able to bind to existing amyloidplaques unlike Ab (1–28) (Lee et al., 1995) and includes residues found by substitution to alterfibril formation (Fraser et al., 1994; Soto et al., 1995; Esler et al., 1996a, b). Transaminationfollowed by SDS-PAGE suggested that residues GIn15 and Lys16 from different monomers wereclose in space. Thus initially peptides labelled with 13C at the carbonyls of residues 15 and 16 weresynthesised. Using a DRAWS (Gregory et al., 1995, 1998) pulse sequence to selectively recouplethese nuclei gave distances of approximately 5 (A for each peptide and the variation of intensitywith mixing time suggested that each 13C had two (or more) equidistant neighbours. This impliesthe completely unexpected result that this peptide forms parallel b-sheet with the residues in exactregister. This was checked by synthesising peptides labelled at Leu 17 and Val 18, which also gavesimilar distances. There remains the question of how these b-sheets interact via their side chainsand what is the ‘‘tertiary’’ fold of the peptide. The many problems of working with insolublepeptides were elegantly side-stepped by synthesising Ab (10–35) with a 3 kDa polyoxyethylene C-terminal extension (Burkoth et al., 1998). This species forms aggregates which resemble those ofthe peptide in forming b-sheet above pH 5.6, binds Congo Red and forms similar ladders ofoligomers on transamination. However, the aggregation processes is reversible and can be studiedin solution by small angle neutron scattering, SANS, and by electron microscopy (Burkoth et al.,1999). The SANS data suggests that the peptide forms rods with a 45 (A radius, so that thediameter is the length of one 26 aminoacid peptide in b-sheet conformation. This is slightly largerthan the protofibrils studied by electron microscopy or AFM (Walsh et al., 1999; Harper et al.,1999). Recently, a more complete analysis of data from both the Ab (10–35) peptide and the PEGderivative (Burkoth et al., 2000) was published. 12 carbonyl–carbonyl DRAWS distancesdistributed along the peptide established that it was almost entirely parallel b-sheet and a modelwas proposed of 6 stacked single b-sheets to a slightly twisted fibril (or protofilament) which mightthen interact with a second fibril to form a double helix.

Recently Antzutkin et al. (2000) reported that multiple quantum 13C NMR measurements onthe Ab (1–40) peptide also strongly favour an in-register structure with parallel b-sheet, inapparent disagreement with conclusions drawn from FTIR spectra. These authors do not argue

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that all amyloid consists of parallel b-sheet, noting that they observed antiparallel b-sheet in theshort Ab (16–22) peptide (Balbach et al., 2000).

The peptide Ab (11–25) has been used by Serpell and Smith (2000) to form fibres which wereexamined by cryo-electron microscopy. The result was that (astonishingly) that it was possible todirectly observe a spacing of 4.7–4.8 (A, corresponding to the spacing between b-sheets. The b-strands run perpendicular to the fibre axis and there was no evidence of twisting of the fibres.Serpell and Smith concluded that ‘‘the b-sheets are arranged in a cylinder or tube, where thestrands are in exact register’’. Fibres formed from the Ab (1–42) peptide showed lower contrast,poorly defined edges and did not appear so straight or rigid as those formed by the Ab (11–25)peptide.

Lansbury et al. (1995) first suggested a relationship between the parallel b-helix architecture andamyloid fibrils noting both the repetitive nature of the parallel b-helix fold and that the b-sheetslay approximately perpendicular to the helix axis. Gay et al. (1991) had earlier shown that a singleleucine-rich repeat of 23 amino acids forms fibrils, suggesting a relationship to the folded LRRstructures (which were unknown at that time). Symmons et al. (1997) explicitly suggested thatthese filaments are composed of parallel b-sheet and that the asparagine in their peptide formedinteractions resembling the asparagine ladder. Lazo and Downing (1997) studied the syntheticpeptides Ac–KLKLKLELELELG–NH2 and Ac–ELELELELELELG–NH2 and suggested fromtheir CD spectra that these peptides formed parallel b-helix structures. However, Khurana andFink (2000) concluded from their FTIR measurements that the conformation of Ac–KLKLKLELELELG–NH2 was different from that of the parallel b-helix proteins and wasprobably a fully extended conformation without turns.

Lazo and Downing (1998) proposed a model for amyloid fibrils inspired by the b-helicalarchitecture but with alternating coils running anti-parallel to accommodate predominantly anti-parallel b-sheet. In fact the model is closer to the lectin fold of concanavalin A (Chothia et al.,1997; Deacon et al., 1997) but has features in common with parallel b-helices such as the short flatsheets with turn regions and thus has the advantage that many sequences can be accommodated.However, stacking of similar residues is excluded because alternate residues would have stronglydisallowed w1 angles and glycine or alanine residues might be required if there are tight turnsbetween the sheets with alternating left- and right-handed a-helical conformations.

Several alternative models of amyloid are briefly reviewed by Li et al. (1999) and compared witha detailed model of Ab (12–42) that they derived from Blake and Serpell’s model, starting from ahairpin of transthyretin. However, their molecular dynamics simulations sharply reduced the twistof the b-sheet, which might be expected as twisted b-sheets can only form all the potentialhydrogen bonds if they are coiled. This is inconsistent with the hydrogen bonds being along thefibril, as suggested by Blake and Serpell (1996). Also the anti-parallel packing of two sheets wouldnormally be associated with untwisted sheets (Chothia et al., 1997). It is possible, as suggested byRochet and Lansbury (2000) that the initial intermediate is untwisted but that twisting occurs onfibril formation. Twisting of a flat structure on fibril formation was observed by Ionescu-Zanettiet al. (1999) at lower resolution by AFM.

A possible model of fibres formed by small peptides might be a single sheet as suggested for Ac–KLKLKLELELELG–NH2 (Khurana and Fink, 2000). Models of fibrils formed from peptideswhich form two or more b-strands can either have a b-meander (up-down) fold so that onepeptide is entirely in one b-sheet, or the peptide can take part in two or more b-sheets. Clearly b-

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meanders such as the model of Li et al. must be anti-parallel. Only parallel or anti-parallel packing(with translations) is possible in the third direction perpendicular to both the peptide and the fibrilin these model, using hydrophobic interactions. As the X-ray fibre diffraction and cryo-electronmicroscopy data suggests that all the b-strands must be approximately perpendicular to the fibrilaxis, other models with hydrogen bonds along the fibril axis will tend to resemble parallel b-helicesor b-rolls but can use either parallel or anti-parallel folding (as can also models of short peptides).These folds do allow anti-parallel packing of side chains as discussed in Section 1.2.5.

Khurana et al. (2001) discuss the binding of Congo Red to a variety of proteins, including fibrilsand b-helical proteins, and suggest that it binds to many different types of structure. However, asCongo Red has its own twofold axis, its binding may suggest that the fibrils and putativeintermediates which bind the dye (Fraser et al., 1994) have a two-fold axis, either because of thesame residues forming anti-parallel b-sheet or by the anti-parallel interaction of protofilaments.Turnell and Finch (1992) experimentally shows how Congo Red can bind by intercalation intoanti-parallel b-sheet in the native insulin structure. By contrast, Li et al. (1999) proposed a CongoRed binding site using only side-chains of their model.

The identification of parallel b-sheet with stacked residues in both Ab (10–35)NH2 and Ab (1–40) and the observation of very flat sheets in the SH3 derived amyloid (Jimenez et al., 1999) andfor the peptide Ab (11–25) studied by Serpell and Smith (2000), suggests that amyloid fibrils, canhave a close relationship to parallel b-helix structures. Flat parallel b-sheets can be stabilised bystacking of similar residues while the only flat anti-parallel sheets known from silk have ratherspecial sequences with alternating glycines although Perutz et al. (1994) predicted a flatpolyglutamine structure. Thus the family 28 hydrolyases of Section 2.1.2 may be the best availablestructural models of a flat b-sheet, irrespective of its topology. Parallel b-helix structures showthat short flat b-sheets can form coils with less regular turn regions and that these turn regionsmay also be stabilised by stacking similar conformations. Whilst making parallel b-helixstructures hard to predict, it allows many peptides to adopt a related structure. The ‘‘advantage’’of the parallel structure is not that stacking is ‘‘better’’ than the interactions across an anti-parallelsheet as there are many interactions giving excellent packing across anti-parallel sheets. However,a peptide can always align with itself in a parallel b-sheet so that valines stack on valines,phenylalanines on phenylalanines and asparagines on asparagines. By contrast, anti-parallelpacking with residue i aligned demands that the sequence has residues i �N and i þN which canpack in a satisfactory manner. Antzutkin et al. (2000) argue that the critical issue is the symmetryof the hydrophobic regions under the 2-fold axis of an anti-parallel b-sheet. When thehydrophobic regions can interact across a 2-fold axis, anti-parallel b-sheet may be favoured.

Clearly in the case of a peptide such as Ab (10–35)NH2 there would be no distinction betweenleft- and right-handed parallel b-helix connections. There is no obvious reason to prefer any of theparallel b-helix or b-roll structures as a starting point for model building although the cryo-EMderived thickness of the SH3 amyloid might suggest a closer relationship to the b-roll, especiallythat seen in pertactin rather than the even thinner structure seen in the alkaline proteases whichrequired rather special sequences of glycines and calcium binding carboxylates (see for a possible model from Jimenez and Saibil).

The size of a parallel b-helix can be measured from the known structures for comparison withlow resolution images from EM or AFM. However, PB1 and PB2 essentially form a sandwich,which is rather similar in dimensions to most other all b-structures. The outward facing b-carbons

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of PB1 and PB2 are about 17 (A apart compared to 15–17 (A for the b-roll region of pertactin. Theunusually narrow b-rolls of the proteases are significantly smaller. The length of the ‘‘sandwich’’in the known parallel b-helix structures is harder to define because of the L shape (Fig. 1).Measuring from the a-carbons of T1 to those at the end of PB3 gives 28 (A but gives only 20 (Ameasuring from the a-carbons of T2 to T3. Using b-carbons again adds 3 (A to the distances butclearly large side chains such as tyrosine can increase these measurements by 10 (A or more evenbefore we add loops. The left-handed hexapeptide repeat structures are triangular prisms of 19 (A(using a-carbons) or 21.5 (A (using b-carbons) along each side and the left-handed antifreezestructure is slightly smaller.

Construction of a detailed model requires data on the role of the side chains interactions tobury hydrophobic surfaces. In the absence of a detailed model it is difficult to predict the effects ofsubstitutions. Prolines are found to inhibit amyloid formation in IAPP (Moriarty and Raleigh,1999) and would tend to destabilise b-sheets, although pectin methylesterase and the receptor forinsulin-like growth factor show that prolines can be accommodated within parallel b-helixstructures. Neighbouring hydrophilic residues might also destabilise hydrophobic packing of b-sheets but could define the positions of turns. Neighbouring residues with the same charge,especially aspartates, would be even harder to accommodate unless these residues are at turns andbind cations or the pH is reduced as suggested by the appearance of fibrils at pH 2.5 in aglutamate substituted peptide (Lazo and Downing, 1999). The effect of the ‘Dutch’ mutation ofE22Q (Watson et al., 1999) is thus consistent with a parallel b-helix model but the increased fibrilformation from the A21G as well as the E22Q mutants (El Agnaf et al., 1998) could arise bydestabilisation of a soluble a-helical form. There is a further complexity in cases where patients areheterozygous for such mutations. Clearly cases such as the E22K mutant described by Bugianiet al. (1998) would allow an alternating pattern of EKEK across a flat b-sheet.

The arguments for a parallel b-helix structure for amyloid fibrils from longer proteins areweaker because the stacking would not be automatically available. The parallel b-helixarchitecture claim to being almost independent of the local sequence is that (1) small or largeinsertions can be introduced every 4–6 residues and (2) that no special motif is necessary except atendency to form b-structure. Experience with protein sequence alignment suggests that frequentgaps with low penalties can give apparently good alignments of unrelated proteins. It is thus notimpossible, but highly speculative, that a large protein could incorporate more than half of itssequence in the stacked flat b-sheets of a parallel b-helix like fibril. However, it should be stressedthat two short peptides, Ab (16–22) and the C-terminal Ab (34–42) form fibrils with an anti-parallel b-sheet. Thus ‘‘similar’ fibrils can apparently form from either parallel or anti-parallelsheet.

5. Conclusions

5.1. Evolutionary relationships

The parallel b-helix fold differs from many enzyme architectures in that it is fundamentallysimple. We can imagine that a gene could be created by copying many times the DNA coding for arelatively short peptide. This is almost certainly the origin of the penta- and hexapeptide repeat

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families of proteins and is the probable origin of all the repetitive folds. If the sequence has a highenough propensity to form b-sheet, stacking of identical residues would favour formation of oneof the folds discussed above, especially as most other folds apparently do not form satisfactorystacks. Similarly high concentrations of a peptide may form amyloid with related b-structures.

Once a parallel b-helix fold has arisen, we can expect it to shrink or expand by duplicating theDNA coding for an entire coil. Again, if there was good stacking in the ancestor, the new coil canbe easily inserted. Thus neither the length of a parallel b-helix nor the distance between featuressuch as an N-terminal a-helix and an active site are ideal tests for homology. We can evenspeculate without evidence that adding a further coil could be useful in binding a longer polymericsubstrate and might be especially likely in proteins binding polysaccharide as the rise per coil(4.8 (A) is comparable to the repeat in a polysaccharide. Clearly the expanded protein mustsubsequently evolve to avoid folding with a coil omitted from the helix and this may be one of themain roles of the N- and C-terminal extensions, which occur in most of the proteins which do notform trimers. This will exert an evolutionary pressure to move away from perfect repetition.

The creation of new repetitive folds has occurred several times during evolution and thus thereis no reason to expect that all the proteins with these folds will be homologous. However, there aresome cases where proteins have evolved from a common ancestor (and some more debatablecases). The left-handed parallel b-helix or hexapeptide repeat family shows homology through (1)close structural similarity; (2) conserved sequence pattern; (3) conserved trimer formation; (4)active sites at similar positions; and (5) in most enzymes a conserved function as an acyltransferase. There are clear sequence similarities enabling us to form seven families within theclass of right-handed parallel b-helix proteins with known L or kidney shaped structures. Theseare (1) the extra-cellular pectate lyase family; (2) the polygalacturonases and rhamnogalactur-onase A; (3) the pectin methylesterases; (4) pectate lyases homologous to PelL; (5) the phage P22tailspike endorhamnosidases; (6) chondroitinase B; and (7) pertactin. The large number ofsimilarities shared by these families arise either from common ancestry or because they arerequired for folding or function. These include (a) the a–L turns (especially at T2 but also at thestart of PB2); (b) the a-helix at the N-terminus of the parallel b-helix forming a coil with a strandof PB2; (c) the a–R turn at the start of PB1; (d) the binding site for substrate is formed by the T3–PB1–T1 region and the polysaccharide substrates bind almost parallel to the parallel b-helix; (e)the long T3 loops tend to be at the N-terminal end of the parallel b-helix and the long T1 loopstend to be at the C-terminal end of the parallel b-helix; (f) all the enzymes degradepolysaccharides, which are closely related except for the chrondroitinase B substrate; and (g)all the identified catalytic residues are carboxylates, arginines and lysines. Some of thesesimilarities are considered in detail below.

(a) T2 turnsThe T2 turns of the parallel b-helix proteins have two unusual features: the angle between the b-

sheets and the detailed form of the turn as discussed in Section 1.2.4. During 1997 a survey of alarge non-redundant database (Oliva et al., 1997) found no other examples of this type of turnbetween b-sheets although a–R turns through similar angles between sheets can be found (forexample residue 107 of concanavalin A (Deacon et al., 1997)). The subsequent occurrence ofsimilar turns in the structure of the insulin-like growth factor receptor (Garrett et al., 1998)suggests that this type of turn may arise by convergent evolution (unless the insulin-like growthfactor receptor is homologous to the pectinases). The only known a–R turns with an external side

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chain in a parallel b-helix are the stack of four in pertactin at T3. However, there is also thepossibility that the sheets approach as in the spiral folds requiring a more complex turn. As theycan stack with or without hydrogen bonds along the helix axis, the a–L (gbE) turns may be anoptimal solution to the problem of turning a b-sheet through 801 and will probably occur in farmore than a quarter of independent folds containing two sheets meeting approximately at rightangles. Thus their occurrence only weakly supports the homology of the right-handed parallelb-helices.

(b) The N-terminal a-helixThe N-terminal a-helix is not required in a right-handed parallel b-helix fold as it does not

occur in pertactin. However, it does solve the problem of capping the parallel b-helix. It may alsobe easier to fit it at the N-terminus rather than insert it after a PB1 strand in place of PB2. Againthe leucine-rich repeat structures show that this type of structure can evolve independently, so thatits occurrence only slightly increases our confidence that all the families except pertactin arehomologous.

(c) The substrate binding siteThe a–R turn at the start of PB1, the substrate binding sites and the position of long T3 and T1

loops are not completely independent. The a–R turn makes a groove which forms the centre of theactive site. The T3 and T1 loops assist in constructing the active site and as PB1 has a twist, it issimpler to have the long T3 loops at the N-terminal end extending the twist of the b-sheet to forma valley (and similarly with the T1 loops at the C-terminal end of PB1). The shape of the coils (orin a case such as pertactin some of the coils) is sufficiently similar that they can be readilysuperposed. This defines one groove as an obvious enzyme active site and binding of substrates orproducts to this site has been observed directly in the extra-cellular pectate lyases, the tailspikeendorhamnosidase and in chrondroitinase B. Polygalacturonases judged by sequence conserva-tion and the effects of site directed mutagenesis and pectin methylesterases simply from sequenceconservation probably use the same site. The pectate lyase PelL also has a conserved cluster ofresidues at the same site. Finally, pertactin has no known enzymatic function and the equivalentsite is not readily accessible.

(f) The substratesAll the right-handed parallel b-helical enzymes degrade polysaccharide and four of the six

enzymes degrade pectin. In fact all four of the sequence families of pectin degrading enzymes withknown structures fold to parallel b-helices. The substrate for the tailspike endorhamnosidasecontains a-linked galactose and rhamnose suggesting some similarity to pectin. Only the b-linkedsubstrate of chrondroitinase B is less closely related and this substrate appears to bind to the samegrove but in the opposite direction. The similarity of the substrates combines with the othersimilarities to argue strongly for the homology of all six enzyme families although the observedbinding of substrate to the pectate lyase PelC via calcium bridges warns us that the details of theenzyme–polysaccharide interactions are not likely to be conserved.

The case of pertactin is open and will remain open until we understand how many differentshapes are possible for a parallel b-helix or find proteins clearly related to both pertactin and thepolysaccharide degrading enzymes. If pertactin does not have a common ancestor with pectinmethylesterase, its N-terminal coils represent an extreme example of convergent evolution.

However, the similarity of the turns at T3 to those of the left-handed family seems almost asmarked even if it is only local.

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Assuming that right-handed parallel b-helix proteins are homologous to each other (note thatthe case of the glutamate synthase domain cannot be discussed until the coordinates are released),the repetitive structure and unusual relationships between the sheets joined by a–L turns maymake the homology unusually obvious. This is because a-helices or b-sheets forming a sandwichcan undergo relative movement during evolution (Chothia and Lesk, 1986) but the sheets in aparallel b-helix are linked together so that the angle at the turn is determined by local packingwhile changes in the size of a side chain in a single coil might require changes in neighbouring coilsif good hydrogen bonding is to be retained. In fact, it is clear from the studies on the tailspikeendorhamnosidase that quite large changes can be introduced by mutagenesis inside the parallelb-helix without significant changes in the positions of the hydrogen bonded main-chain atoms(Schuler et al., 2000).

More distant relationships between the right-handed parallel b-helix proteins and the otherfolds are possible but do not seem well supported. There may be an evolutionary path from theleucine-rich repeat proteins to the right-handed parallel b-helix enzymes but we do not need topostulate one if stacking can easily generate new repetitive folds when DNA is duplicated. Thespiral folds are apparently unrelated to right-handed parallel b-helix enzymes because it isimpossible to simultaneous superpose the two sheets despite the angle between them being similar.

5.2. Future directions

The most obvious future developments in the structural biology of the parallel b-helix enzymeswill be the detailed characterisation of their substrate binding as this defines specificity, which inturn should be at least partially understood if these enzymes are to be used in biotechnology. Forexample, the properties of pectin both in plant tissue and in food depend, not only on its degree ofesterification, but also on the pattern of esterification. The patterns of esterification produced bythe various pectin methylesterases are different, which will only be understood once we know themolecular details of substrate binding to pectin methylesterases. Comparison of the substratebinding sites of the different enzyme families may also suggest how the righthanded parallel b-helix enzymes have evolved from a common ancestor.

The second development will be the determination of further structures with parallel b-helixfolds, some of which will have no obvious sequence relationships to the known structures.Experimental tests of the predicted structures of the pentapeptide repeat family (Bateman et al.,1998) and the Pseudomonas syringae ice-nucleation protein (Graether and Jia, 2001) are eagerlyawaited but each new structure will help us to understand the fold and eventually to predict if asequence will fold to either a right- or left-handed parallel b-helix.

In addition to calculations using the packing of side chains to determine which sequences willfavour the different chiralities, it may be possible to test experimentally by protein engineeringwhether a left-handed parallel b-helix can be converted into a right-handed fold. One approachmight be to replace the chiral isoleucines in a left-handed structure by leucines and methionines.However, if an active enzyme is to be designed it will also be necessary to reverse the order of thecoils in the sequence and then search for mutants restoring folding and activity.

Structure prediction is clearly advancing rapidly using mostly using ‘‘threading’’ (Sippl andFlockner, 1996; Jones et al., 1999). The increased number of known structures allows the use ofeach as a search model but this approach might not recognise new folds which formed parallel b-

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helices such as is suggested for the pentapeptide repeats. However, it will certainly now be possibleto use the short leucine-rich repeat structures as alternatives to the parallel b-helix proteins asmodels for proteins with 20–24 residues per coil. It might also be possible to use our current anddeveloping understanding of both folds as a screen after a possible model has been built. Forexample, examination of the known structures suggests that any parallel b-helix model withoutextensive stacking of residues is likely to be wrong.

The prediction of new repetitive folds is likely to advance by incorporating more explicitlythe repetitive nature of the parallel b-helix. Examination of the recently determinedstructures of several right-handed parallel b-helix proteins without obvious sequencehomology suggests that no single sequence profile is likely to find all these structures. Forexample amongst the pectinases, aromatic stacks are found on PB3 in the pectate and pectinlyases, PB2 in the esterases and PB1 in PelL while the hydrolases have few aromatic stacks.Thus one possibility is to search for the occurrence of stacks of similar residues insuccessive coils rather than for a single pattern. The variable lengths of the turn regions hasso far hindered the development of such prediction methods and the observation byBateman et al. (1998) that there is no obvious periodic signal in the sequences of theleft-handed parallel b-helix family corresponding to a coil also shows the problemsneeding to be overcome. However, the identification of the antifreeze protein fromTenebrid molitor as b-helical (Liou et al., 1999) and the recent identification of twopectinases as b-helical by automatic servers shows that prediction of b-helical structures fromthe sequence is frequently possible.


We thank Alex Bateman, Alex Drake, Curtis Johnson, Peter Lansbury and Bill Turnell forhelpful discussions, Paul Emsley and Neil Isaacs, Fran Jurnak, Leping Li, Lee Darden, Bill Kayand Peter Davies for sending coordinates before their general release, and Bostran Kobe, MirekCygler, Peter Lansbury and Jean-Christophe Rochet for preprints of their articles. The figuresabove were made with the programs MOLSCRIPT (Kraulis, 1991) or MOLMOL (Koradi et al.,1996). This work was funded by the BBSRC (UK) and EU projects AIR2-CT94-1345 and BIO4-CT96-0685.


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