Crystal Structure of Fungal LectinSIX-BLADED �-PROPELLER FOLD AND NOVEL FUCOSE RECOGNITION MODE FORALEURIA AURANTIA LECTIN*

Received for publication, March 14, 2003, and in revised form, April 28, 2003Published, JBC Papers in Press, May 5, 2003, DOI 10.1074/jbc.M302642200

Michaela Wimmerova‡§¶, Edward Mitchell¶�, Jean-Frederic Sanchez**‡‡, Catherine Gautier**,and Anne Imberty**§§

From the ‡National Centre for Biomolecular Research and Department of Biochemistry, Masaryk University, 611 37 Brno,Czech Republic, �European Synchroton Radiation Facility Experiments Division, BP 220, F-38043 Grenoble cedex, France,and **Centre de Recherches sur les Macromolecules Vegetales-CNRS (affiliated with Universite Joseph Fourier),BP 53, F-38041 Grenoble cedex 09, France

Aleuria aurantia lectin is a fungal protein composedof two identical 312-amino acid subunits that specifi-cally recognizes fucosylated glycans. The crystal struc-ture of the lectin complexed with fucose reveals thateach monomer consists of a six-bladed �-propeller foldand of a small antiparallel two-stranded �-sheet thatplays a role in dimerization. Five fucose residues werelocated in binding pockets between the adjacent propel-ler blades. Due to repeats in the amino acid sequence,there are strong similarities between the sites. Oxygenatoms O-3, O-4, and O-5 of fucose are involved in hydro-gen bonds with side chains of amino acids conserved inall repeats, whereas O-1 and O-2 interact with a largenumber of water molecules. The nonpolar face of eachfucose residue is stacked against the aromatic ring of aTrp or Tyr amino acid, and the methyl group is locatedin a highly hydrophobic pocket. Depending on the pre-cise binding site geometry, the �- or �-anomer of thefucose ligand is observed bound in the crystal. Surfaceplasmon resonance experiments conducted on a seriesof oligosaccharides confirm the broad specificity of thelectin, with a slight preference for �Fuc1–2Gal disaccha-ride. This multivalent carbohydrate recognition fold is anew prototype of lectins that is proposed to be involvedin the host recognition strategy of several pathogenicorganisms including not only the fungi Aspergillusbut also the phytopathogenic bacterium Ralstoniasolanacearum.

Lectins are carbohydrate-specific proteins that are keyplayers in many recognition events at the molecular or cel-lular level (1). Fungi, either mushrooms or filamentous fungi,

often depend on host association (symbiosis or parasitism)and appear to use lectins for host recognition and/or adhe-sion. One of the first examples of a lectin-mediated interac-tion between a fungus and its host was discovered in thenematode-trapping fungus Arthrobotrys oligospora (2). Inhigher fungi, lectins are involved in molecular recognitionduring the early stage of mycorrhization. An example is theirrole in the high specificity of the Lactaria mushroom/treesymbiotic association (3). A role of lectins in mycoparasitismhas been proposed for a number of human pathogens such asCandida albicans (4), the agent causing oral candidosis, andAspergillus fumigatus (5), which is a major life-threateningpathogen in hospital environments, responsible for invasivepulmonary aspergillosis in immunodeficient patients (6).Lectin-mediated recognition is also involved in plant myco-parasitism (7, 8).

Due to the importance of their biological role, there is in-creasing interest in fungal lectins. However, there is only lim-ited information about them, and although several crystalshave been obtained, including the lectins from Flammulinaveltipes (9), Pleurotus cornicopiae (10), Pleurotus ostreatus (11),Sclerotium rosfii (12), and Aleuria aurantia (13, 14), no crystalstructure has yet been determined.

The lectin from the orange peel mushroom, A. aurantia(AAL),1 has been purified from the fruiting bodies of thefungus as a 72-kDa protein composed of two identical sub-units and has been shown to exhibit millimolar range affinity(Kd � 1.6 � 10�4 M) for fucose (15). Later, the primarysequence was determined and demonstrated the presence ofsix internal repeats of about 50 amino acids (16). Cloning ofthe gene allowed production of the recombinant lectin inEscherichia coli (17). Further characterization of the lectinspecificity demonstrated that all fucose-containing disaccha-rides present on glycoconjugates (�Fuc1–2Gal, �Fuc1–3GlcNAc, �Fuc1–4GlcNAc, and �Fuc1–6GlcNAc) displayedsimilar binding to the lectin, higher than that shown forhighly branched oligosaccharides such as the determinants ofLewis histo-blood groups (15, 18, 19). Since AAL is the onlyavailable lectin with high affinity for the �Fuc1–6GlcNAcpresent in the core of complex N-glycans, it is widely used inthe fractionation of glycoproteins.

L-Fucose, as a component of cell surface complex oligosaccha-rides, is a key participant for cell surface recognition. Never-theless, until very recently, no characterization of any fucose-lectin crystal structure was attained. In the last year, the

* Travels and visits between the National Center for BiomolecularResearch and Centre de Recherches sur les Macromolecules Vegetalesare supported by a BARRANDE exchange program. The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1OFZ) have beendeposited in the Protein Data Bank, Research Collaboratory for Struc-tural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

§ Stay in Grenoble supported by the French minister program forinvited scientists and partial financial support from the Ministry ofEducation of the Czech Republic by Grant LN00A016.

¶ These two authors contributed equally to this work.‡‡ Supported by a grant from the French association La Ligue Contre

le Cancer.§§ To whom correspondence should be addressed. Tel.: 33-476-03-76-

36; Fax: 33-476-54-72-03; E-mail: imberty@cermav.cnrs.fr.

1 The abbreviations used are: AAL, A. aurantia lectin; r.m.s., rootmean square.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 29, Issue of July 18, pp. 27059–27067, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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structure of fucose binding lectins from legume plant Ulexeuropaeus (lectin I) (20), from animal Anguilla anguilla (eel)(agglutinin) (21), and from bacterium Pseudomonas aeruginosa(lectin II) (22) have been solved with ligated fucose. Interest-ingly, these three different proteins display different bindingmodes toward the same monosaccharide.

The crystal structure of AAL-fucose complex reported herehas no similarity to any other described fucose-binding lectin.It represents a new fold present in a lectin family common toseveral pathogenic bacteria and fungi.

EXPERIMENTAL PROCEDURES

Materials—AAL was purchased form from Vector Laboratories, Inc.(Burlingame, CA). The sample was dissolved in water, and salts wereremoved by ultracentrifugation using Nanosep 3K Omega (Pall Corp.,Ann Arbor, MI) with washing by water. Lewis a (�-L-Fuc-(134)[�-D-Gal-(133)]-D-GlcNAc), Lewis X (�-l-Fuc-(133)[�-D-Gal-(134)]-D-Glc-NAc), blood group A (�-L-Fuc-(132)-[�-D-GalNac-(133)]-D-Gal), bloodgroup B (�-L-Fuc-(132)-[�-D-Gal-(133)]-D-Gal), and blood group H typeII (�-L-Fuc-(132)-�-D-Gal-(134)-D-GlcNAc) trisaccharides were pur-chased from Dextra Laboratories (Reading, UK). L-fucose, p-nitrophe-nyl-�-L-fucoside, p-nitrophenyl-�-L-fucoside, �-L-Fuc-(132)-D-Gal, and�-L-Fuc-(132)-�-D-Gal-(134)-D-Glc were purchased from Sigma.

Crystallization and Data Collection—Crystallization trials were per-formed with Hampton crystallization screens I and II (Hampton Re-search, Laguna Niguel, CA) using the hanging drop technique. Crystalswere obtained using the following conditions: 2 �l of precipitant (0.1 M

sodium cacodylate buffer, pH 6.5, 0.2 M magnesium acetate tetrahy-drate, 20% polyethylene glycol 8000) mixed with a solution of 2 �l ofAAL at a concentration of 10 mg/ml and L-fucose at a concentration of137 �g/ml. The drops were allowed to equilibrate over a reservoir of 1 mlof the precipitating solution at 20 °C. Crystals grew as platelets tomaximum dimensions of 0.3 � 0.3 � 0.05 mm3 after 1 week. Theybelong to space group P21 with unit cell dimensions of a � 45.97 Å, b �86.41 Å, c � 77.85 Å, and � � 90.62° at 100 K. The asymmetric unitaccommodates two monomers, corresponding to a Vm of 2.20 Å3 Da�1

and a solvent content of 46% solvent. A mercury derivative was pre-pared by soaking a crystal in 1 mM sodium ethylmercurithiosalicylate(thimerosal) (Hampton Research) for 24 h.

Crystals were cryo-cooled at 100 K after soaking them in eitherparaffin oil or 30% (v/v) glycerol in precipitant solution for the nativeand mercury derivative, respectively. All data images were recorded onan ADSC Q4R CCD detector (Quantum Corp.) on the fixed energy

beamline ID14–1 at the ESRF (Grenoble, France). Single wavelengthhighly redundant anomalous diffraction data to 2.0-Å resolution werecollected from the thimerosal-soaked crystal and native data to 1.5-Åresolution. Measurements were made at a single x-ray wavelength of0.934 Å. Diffraction images were processed using MOSFLM (23) andscaled and truncated to structure factors using the CCP4 (24) programsSCALA and TRUNCATE. Data processing statistics are presented inTable I.

Structure Determination—The crystal structure was solved using thesingle wavelength highly redundant anomalous diffraction techniquewith data from the mercury derivative. Harker sections of the anoma-lous difference Patterson map showed two peaks corresponding to onemercury per monomer in the asymmetric unit. Initial mercury sitecoordinates, phasing, and solvent flattening were carried out with au-toSHARP (25),2 which located the two mercury sites. autoSHARP wasalso directed to search for noncrystallographic symmetry, and the re-sulting matrix was further refined, together with averaging and phaseextension, using DM (27) to give an electron density map of excellentquality. An initial structure was built automatically using ARP/wARP(28), and side chains were docked to give 474 residues out of a total of624 for the asymmetric unit cell. Manual building using O (29) gave amore complete model, which was then used as a search probe forAMORE (30) molecular replacement using the nonisomorphous highresolution native data.

AMORE gave two clear solutions, which were used to generate a newnoncrystallographic symmetry averaging matrix. Phase extension, av-eraging, and solvent flattening with DM generated new phases andfigures of merit for the native data, which was followed by a completeautomatic construction, side chain docking, and an initial water mole-cule construction with ARP/wARP, which gave a model of 588 residuesout of 624 with an Rcrys of 20.3% R and Rfree of 23.7%. The remainder ofthe residues and the fucose molecules clearly defined in density werepositioned manually using O. Further refinement cycles with REF-MAC, including automatic water molecule placement using ARP/wARP,manual rebuilding with O, and construction of alternative conforma-

2 C. Vonrhein, E. Blanc, P. Roversi, and G. Bricogne, manuscript inpreparation.

TABLE IData collection and phasing statistics

Values in parenthesis refer to the highest resolution shell. Rmerge � ��I ��I��/¥�I�, where I � observed intensity. Rcullis(anom) � r.m.s. (DANOcalc �DANOobs)/r.m.s. (DANOobs). Phasing power(anom) � r.m.s. (DANOcalc)/r.m.s.(DANOcalc � DANOobs).

Mercury Native

Beamline ID14–1:ESRF ID14–1:ESRFSpace group P21 P21Resolution (Å) 2.0 1.5Highest resolution shell (Å) 2.07–2.00 1.55–1.50Wavelength (Å) 0.934 0.934Cell dimensionsa (Å) 46.9 46.0b (Å) 86.2 86.4c (Å) 78.2 77.8� (degrees) 90.0 90.0� (degrees) 94.2 90.6� (degrees) 90.0 90.0Total number of hkl 615086 534572Number of unique hkl 41982 94102Average multiplicity 14.7 (15.0) 3.6 (2.8)Rmerge (%) 8.6 (20.0) 4.9 (37.4)Average I/�(I) 6.9 (3.3) 8.4 (1.9)Completeness (%) 99.9 (99.9) 96.8 (96.8)Completeness for anomalous

data (%)99.9 (99.9)

Wilson B-factor (Å2) 11.9 14.4Phasing power (anom) to 2.0 Å 1.67Rcullis (anom) to 2.0 Å 0.77Average figure of merit to 2.0

Å0.36

TABLE IIRefinement statistics

Parameters Values

Amino acids 2 � 312Protein atoms 2436 A; 2457BSolvent atoms 1166Sugar atoms 2 � 5 � 11Resolution limits (Å) 19.96-1.50Working set R (observation) 0.144 (92,218)Test set R (observation) 0.179 (1884)Highest resolution shell

Working set R 0.263 (5239)Test set R 0.273 (107)Cruickshank’s DPI 0.067

Average Biso (Å2)All atoms 12.8Protein atoms A: 12.1, B: 16.7Solvent 32.8

Residues with incompletechain density

A: Ala254, Lys296. B: Asp42,Gln51, Glu56, Lys69, Gln101,Lys108, Lys136, Lys152,Val163, Lys188

Residues in alternativeconformations

A: Arg21, Leu59, Val81, Ser88,Val109, Ser150, Ser207,FUC1002.

B: Ser14, Val25, Leu59, Ser95,Thr154, Asn197, Ser205,Ser207, Ser283, Gln289,Gln291, FUC1002.

Number of outliers onRamachandran plota

0

Overall G factora 0.0Distance deviations

Bond distances (Å) 0.013Bond angles (degrees) 1.556Planar groups (Å) 0.008Chiral volume

deviation (Å3)0.165

a Analyses were determined by PROCHECK (26).

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FIG. 1. A, ribbon diagram of monomer A of AAL complexed with fucose shown as sticks. B, Connolly surface of AAL color-coded according to thehydrophobicity potential (from brown for hydrophobic to blue for hydrophilic) displayed with the MOLCAD program (50). The red balls representthe water molecules present in the tunnel. C, dimer of AAL with stick representation of the amino acids involved in the interaction of monomers.D, amino acid sequence of AAL shown as six aligned repeats. The four �-strands are indicated by arrows. Residues with a colored background areinvolved in the fucose binding sites. They are color-coded according to their blade as in A and are underlined when they participate in binding offucose in site i � 1. Residues in red participate in the dimer interface. Residues in italic type line the central cavity of the monomer. Boxed residuesare hydrophobic amino acids that make van der Waals contacts between adjacent blades. E, stereoview of site 1 in monomer A with the final�-weighted 2Fo � Fc electron density map around the fucose molecule. The density is contoured at 1.0 �. All molecular drawings in all figures wereprepared with MOLSCRIPT (51) and RASTER-3D (52) unless otherwise indicated.

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tions, where necessary (with occupancies estimated from the refinedrelative B-factors of the conformations), resulted in a final model of all624 residues with 10 fucose molecules (5 bound per monomer) and 1166water molecules with an Rcrys of 14.4% and Rfree of 17.9% to 1.5-Åresolution. Side chain atoms not defined in electron density were re-tained in the model but with an occupancy set to zero (Table II).

Surface Plasmon Resonance Measurements—All surface plasmonresonance experiments were performed with a Biacore 3000 (BiacoreAB, Uppsala, Sweden) at 25 °C using HBS buffer (10 mM Hepes, 150 mM

NaCl, pH 7.4) and a flow rate of 5 �l/min. Measurements were carriedout simultaneously on all four measuring channels using three differentconcentrations of immobilized AAL, whereas the fourth channel wasused as the control flow cell. A research grade CM5 sensor chip wasactivated with a 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide solution for 10 min, and 50 �l of AAL in 5 mM

maleate buffer, pH 7.0, at concentrations of 50, 10, and 2 �g/ml respec-tively, was injected to a particular flow cell. The unreacted species onthe sensor surface were blocked by 1 M ethanolamine. The blank chan-nel was treated identically except for the lectin injection.

30 �l of carbohydrate solutions (concentrations between 0.39 and 200�M) in running buffer were injected into the flow cells using the kinjectmode. The equilibrium response (after subtraction from the response ofthe reference surface) of each experiment was used to create curves ofanalyte binding, which were fitted to a 1:1 steady-state affinity modelusing Origin version 6.1 software (OriginLab Corp.).

RESULTS

AAL Overall Fold—The six tandem repeats of the AALamino acid sequence are organized around a six-foldpseudoaxis of symmetry (Fig. 1A). Each repeat consists of atwisted four-stranded antiparallel �-sheet, and the overall ar-rangement corresponds to the fold previously described as asix-bladed �-propeller (31, 32). The global shape is a shortcylinder, or tore, with an approximate diameter of 45 Å and aheight of 35 Å. In the �-propeller fold, each consecutive �-sheethas its first strand (number 1) lining the central cavity of theprotein and the last one (number 4) most exposed to solvent at

the cylinder surface. Loops connecting the strands within eachmodule are rather short, with the exception of blade III (aminoacids 108–162) that displays longer loops (Fig. 1D). The con-secutive blades are connected by long segments that run fromthe outside of the protein to the central tunnel. Superposition ofthe main chain �-strand repeats gives r.m.s. values between1.3 and 1.4 Å, indicative of their high spatial similarity.

Several amino acids at the N and C termini of the peptidechains protrude from the base of the �-propeller cylinder, as-sociating in a small antiparallel two-stranded �-sheet thatforms a separated domain.

The inner cavity of AAL has a tunnel shape with a diameterof about 8 Å in its middle part and almost closed off on the Nterminus side of the first inner �-strands (Fig. 1B). This cavityhas a strong hydrophobic character, being formed mostly by theconserved alanine residues of the first strands of each propellerblade. The core is filled with a set of about 50 water moleculesforming a well ordered hydrogen bond network (average B-factor value of 12.5).

Oligomeric State—AAL has been described as a dimer insolution (15) and is also observed as a dimer in the crystalstructure. The two monomers are very similar, and superim-position of their backbones gives an r.m.s. value of 0.26 Å. Apseudo-2-fold axis of symmetry generates this dimer in thecrystal (Fig. 1C). The small domain created by the antiparallelassociation of the N-terminal and C-terminal peptides plays akey role in the dimerization, additional contact being mediatedby four loops (those interconnecting blades I and II and bladesII and III and the loops between strands �2-I and �3-I andbetween strands �2-II and �3-II). Hydrophobic contacts involvethe C terminus Trp312 from each monomer with Lys83 from theother. In addition, one tyrosine residue, Tyr6, interacts viaaromatic ring stacking with its counterpart on the other mon-

TABLE IIIContacts (distances in Å) between fucose and monomer A (roman characters) and monomer B (italic characters)

Conserved hydrogen bonds and hydrophobic contacts are in boldface type. The maximal error on non bonding distances is 0.13 Å as evaluatedfrom Cruickshank’s DPI value based on the R factor (Table II).

Ligand atom Site 1 Site 2 Site 3 Site 4 Site 5

O-1� W1 (2.7/2.9) W1 (2.6/3.0) W1 (2.7/3.0) W2 (2.7)W2 (2.7/2.5) W3 (3.1/2.9) W3 (3.1) W2 (2.8) (Ser104.O - 3.1)a

O-1� Gln101.OE1 (3.2/2.6) Wat1 (2.7/2.7)W1 (3.1/3.1) Wat3 (3.1)

Wat7 (3.2/3.0) (bridge)b

O-2 Gln101.N (3.0/3.1)c Gly203.N (2.8/2.9)W5 (2.8) W4 (2.8) Gln101.OE1 (2.8/3.3)c W3 (2.6/3.1) W3 (2.7/2.7) W4 (2.7/2.9)

Wat2 (2.7/2.9)c W5 (2.8/3.0) W5 (2.9) W5 (2.6)W6 (2.8/3.0) (Gln72.OE1–2.75)d

O-3 Glu36.OE1 (2.7/2.7) Glu89.OE1 (2.6/2.7)c Glu146.OE1 (2.6/2.6) Glu191.OE1 (2.6/2.6) Glu238.OE1 (2.6/2.6)Trp97.NE1 (3.0/3.0) Trp153.NE1 (2.8/2.7)c Trp199.NE1 (2.9/3.0) Trp245.NE1 (2.9/2.9) Trp298.NE1 (2.9/2.9)W6 (3.0/2.9) W6 (2.9/3.1) W6 (2.8/2.8) W6 (2.8/2.9)

O-4 Arg24.NE (2.8/2.8) Arg77.NE (3.0/3.0)c Arg131.NE (2.8/2.8) Arg179.NE (2.8/2.9) Arg226.NE (2.8/2.8Glu36.OE2 (2.6/2.7) Glu89.OE2 (2.7/2.7)c Glu146.OE2 (2.7/2.7) Glu191.OE2 (2.7/2.6) Glu238.OE2 (2.6/2.5)

O-5 Arg24.NH2 (2.9/2.9) Arg77.NH2 (2.9/3.1)c Arg131.NH2 (2.9/2.9) Arg179.NH2 (2.8/2.9) Arg226.NH2 (2.9/2.9)

Hydrophobic(C-4, C-5,C-6)

Trp15 Trp68, Trp120 Ile173 Trp219

Ile74 Pro128 Pro223 Ile274

Ile76 Ile130 Leu178 Ile225 Ile276

Tyr92 Trp149 Trp194 Tyr241 Trp292

C-1, C-2 Val91 Cys193

a In monomer A only: hydrogen bond with A chain related by symmetry 1 � x, y � 1/2, 2 � z.b Values averaged over the �-Fuc and �-Fuc in the site (less than 0.02-Å deviation).c In monomer B only: hydrogen bond with B chain related by symmetry x, y, z � 1.d Water molecule bridging to Tyr241.OH.

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omer through the 2-fold axis. Four main hydrogen bonds arealso established between the side chains of Asp263 and Ser283

and between the Trp312 nitrogen side chain and backbone car-bonyl backbone of Leu59.

This dimerization mode creates a “back to back” associationof the two cylinder-shaped monomers. It closes the internalcavities on the N terminus side of the inner strand but leavesthe other side of the cavity (i.e. the most open one) accessible tosolvent. The fucose binding sites are exposed on each side of thedimer, at distances ranging from 50 to 70 Å from each other.

Fucose Binding Sites—The crystal structure of the complexbetween AAL and fucose reveals five fucose residues bound permonomer (Fig. 1, A and B). These are located between consec-utive blades, in binding sites consisting of pockets at the exter-nal face of the cylinder (Fig. 1E). For simplicity, the site locatedbetween blades I and II will be named site I, and the followingones will be named consecutively. The site between blades VIand I, which would have been referred as site VI, does notcontain any electron density corresponding to a bound fucosemolecule. The contacts observed in the five sites of monomer A

and monomer B are listed in Table III. The two monomers arealmost equivalent, with the exception of marginal contact withsymmetry-derived monomers in site IV of A and site V of B.Therefore, only the sites of monomer A are described morelengthily and shown in Fig. 2.

The five fucose binding sites are not equivalent, but theyhave the same architecture and present invariant features.They are made up in the crevasse between two adjacent blades,and it is mostly amino acids of the four strands (rather than theloops) in each blade that participate in binding. As displayed inthe alignment of sequence repeats in Fig. 1D, amino acids ofblade i can therefore participate in site i or site i � 1. Con-served features of the binding sites consist of five hydrogenbonds between fucose and protein (Fig. 2); for fucose in site i,they involve the side chain of three highly conserved residues,Arg of strand �2 � i, Glu of strand �3 � i and Trp of strand�4 � (i � 1). These five hydrogen bonds make a compactnetwork, the geometry of which is strictly conserved in the fivebinding sites. In addition to NH2 of Arg donating a hydrogenbond to the fucose ring oxygen, two cooperative sets of bonds

FIG. 2. Stereoscopic representationof the fucose binding sites I, II, andIV of monomer A. Hydrogen bonds aredisplayed as dashed lines.

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are created; NE1 of the site Trp gives a hydrogen bond to O-3that in turn donates a hydrogen bond to one acidic oxygen ofthe site Glu, whereas NE of Arg is bridged to the other oxygenof Glu via OH-4 of the fucose.

The second part of the binding site is characterized by ahydrophobic region. The closer and most conserved hydropho-bic contact involves a Trp/Tyr residue at the extremity of �4 �(i � 1). The aromatic ring stacks against the flat nonpolar faceof fucose, with contacts with the methine and methyl groups atC-4, C-5, and C-6. The conserved isoleucine residue of strand�2 � (i � 1) establishes additional hydrophobic contact with themethyl group at C-6 of fucose. Longer distance contact involvesthe conserved Trp of strand �1 � i.

The interactions described above leave hydroxyl groups O-1and O-2 exposed to solvent. In all sites, fucose interacts with anumber of water molecules that varies between two and five.The mean hydration number of fucose is four, which is anunusually high value for a monosaccharide in a protein bindingsite. Analysis of the water molecules hydrogen-bound to thesugar indicates that they are not randomly scattered but can beclustered in different sites. Fig. 3 illustrates the seven sitesthat have been identified. W4 occupies roughly the position ofbackbone NH in the sites where the connecting loop betweenblades does not come into contact with the fucose. Interest-ingly, almost none of these water molecules establish hydrogenbond directly to the protein but are rather in contact with otherwater molecules from the solvent. The only exception, W7,stabilizes the �-anomer in site IV (see below), by bridging theO-1a to the OH of Tyr241 in both A and B monomers.

Since the six �-propeller blades are not identical, there aresome differences between the five binding sites (Fig. 2). First,fucose is not bound in the same configuration in the differentsites. In both monomers, the sugar in binding sites I, III, and Vadopts the �-configuration (equatorial O-1), whereas it isbound in the �-configuration (axial O-1) in binding site IV. Inbinding site II, both anomers can be identified in the electrondensity with an �/� population of 35/65 and 55/45 in monomersA and B, respectively. This selection of anomeric configurationcan be correlated to differences in the architecture of the bind-ing sites. The number of hydrogen bonds between fucose andprotein varies from five to seven: five in sites I, III, and V; sixin site IV; and seven in site II. When looking at the differencesbetween individual binding sites, it appears that I, III, and Vare very similar. In these three sites, hydroxyl groups O-1 andO-2 of the fucose ligand are exposed to the solvent and do notparticipate in binding to the protein. On the other hand, inbinding sites II and IV, the outermost external �-strand of theblade is oriented slightly differentially, and the beginning ofthe interblade connecting loops is in contact with fucose. Thisresults in hydrogen bonds between the hydroxyl group at O-2and the backbone amide nitrogen of Gln101 and Gly203 in sitesII and IV, respectively. In site II, Gln101 also interacts via itsside chain, resulting in a deeper binding site.

In aqueous solution, the two configurations of fucose ex-change freely by tautomerization. In the crystal, the �-anomeris fixed in three binding sites, and the �-anomer is fixed in one.It has been checked that this selection does not arise fromsteric hindrance and that both anomers could fit in all sites. Itseems more likely that the complex network of water moleculesand the presence or absence of the contacting loop at O-2influence the selection.

Analysis of the crevasse between blade VI and blade I, wherefucose does not bind, indicates that there is indeed a pocketbetween the two blades. Nevertheless, two of the polar aminoacids responsible for hydrogen bonding of fucose are missing:Ser277 instead of Arg and Gln299 instead of Glu in blade VI.

Furthermore, the aromatic amino acid that should stackagainst the apolar face of fucose is replaced by Arg39 in blade I(Fig. 1D).

Our crystallographic evidence of five fucoses bound on eachAAL monomer is in contradiction to previous biochemical stud-ies performed by equilibrium dialysis that concluded that onlyone carbohydrate binding site existed per monomer (15). Thediscrepancy between stoichiometry values obtained by equilib-rium dialysis and those determined by crystallography may bedue to the nonequivalence of the five sites. It seems likely thatone of them, most certainly site II that establishes seven hy-drogen bonds with fucose, has a higher affinity for fucose thanthe other ones.

AAL Oligosaccharide Specificity—The particularity of AAL,when compared with other fucose binding lectins, is its largerange of affinity. Contrary to U. europaeus agglutinin isolectin1 or A. anguilla agglutinin that have a strong preference for�Fuc1–2Gal terminal disaccharides, AAL binds oligosaccha-rides or glycoconjugates bearing �Fuc linked in the 1–3, 1–4,and 1–6 positions all equally well (15, 18). In fact, the relativelyhigh affinity for fucose measured by equilibrium dialysis (Kd �16 �M) (15) or by surface plasmon resonance experiments (Kd �33 �M) (33) is not further increased when various fucose-con-taining disaccharides are tested (19). A high resolution NMRstudy of free and AAL-bound �Fuc1–6�GlcNAc-O-Me demon-strated that only the fucose is bound in the protein binding site,whereas the GlcNAc moiety rotates freely in the bulk solvent(34).

It therefore appears that for disaccharides, only the terminalfucose is establishing contact with the proteins. Since A. an-guilla agglutinin shows higher affinity to large fucose-contain-ing glycoproteins such as human lactotransferrin (18), it is ofinterest to test which of the fucose-containing oligosaccharidescould be recognized.

A surface plasmon resonance binding assay was used todetermine equilibrium dissociation constants (KD) for AALbinding of some fucose-containing saccharides. Fig. 4 showstypical sensorgrams obtained after the injection of analytesover the lectin-covered surface. Since association and dissocia-tion phases were rapid, binding curves for all substrates werecalculated using the steady-state parts of experimental curves.KD values were investigated using Scatchard plot analysis andby fitting the data to a saturation curves. The results are

FIG. 3. Superposition of all 10 binding sites, with only onefucose represented. The seven clusters of water molecules have beenindicated.

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summarized in Table IV. Final values were obtained by curvesfitting using Origin software version 6.1, which enabled simul-taneous evaluation of all three curves for each substrate withshared KD. The calculated constants were in agreement withvalues obtained by linearization methods. A comparison of KD

values derived from surface plasmon resonance experimentsreveals that AAL lectin shows a slightly higher affinity todisaccharide �-L-Fuc-�-D-Gal than to L-fucose and Lewis trisac-charides. These results are in agreement with previously pub-lished data (19), and equilibrium dissociation constants for allmeasured sugars are within 1 order of magnitude, demonstrat-ing that AAL preferentially recognizes a fucose moiety.

The presence of an aromatic group at position O-1, either inthe � or � configuration, lowers the affinity, which is unusualfor protein-carbohydrate interactions, which are often charac-terized by a hydrophobic patch close to the binding site. Whenlooking in detail at all of the �Fuc1–2Gal-containing oligosac-charides tested, it appears that the presence of another sugarat position 3 of Gal (i.e. blood group A and B trisaccharides)does not affect the affinity, whereas substitution at position 1 ofGal (i.e. fucosyllactose, blood group H type II, and Lewis oligo-saccharides) results in a decrease of the affinity, suggesting asteric hindrance in this region.

DISCUSSION

Comparison with Other �-Propeller Proteins—The modularorganization of �-propeller proteins can create symmetry rang-ing from 4- to 8-fold. They have a cylindrical/disk shape incommon and a high structural rigidity. The �-propellers haveextreme diversity in function and are found in many organismssuch as viruses, bacteria, and eukaryotes (31, 32, 35). For agiven number of blades, there are no sequence similaritiesbetween the different members of the family. Interestingly, a

rather high number of �-propellers with known crystal struc-tures are carbohydrate-active enzymes, such as the five-fold�-L-arabinanase (36), the six-fold sialidase/neuraminidase (37)and glucose dehydrogenase (38), and the seven-fold galactoseoxidase (39). The only lectin that has been identified as a�-propeller is tachylectin-2 (40). It consists of five blades withhighly similar repeats and five GlcNAc-binding sites locatedbetween the blades. When comparing overall structures andcarbohydrate binding sites, there are no similarities betweenAAL and tachylectin-2 (Fig. 5). The overall shape of tachylec-tin-2 is disklike, and the amino acids involved in the carbohy-drate binding sites are located in long loops and not in�-strands as in AAL. It therefore seems that there is no phy-logenic relationship between these two lectins.

The DALI program (41) was used to identify proteins withclose structural similarities available in the Protein Data Bank(42). The highest scoring hits were then structurally alignedwith the structure comparison tool of the Proceryon software(ProCeryon Biosciences). Structures most similar to AAL aresialidases of various origins. The bacterial Salmonella typhi-murium LT2 neuraminidase (43) (Protein Data Bank code2SIM) and Micromonospora viridifaciens sialidase (44) (Pro-tein Data Bank code 1EUT) superimpose on the AAL mainchain C-� coordinates with r.m.s. values of 2.35 Å for 212 aminoacids and 2.38 Å for 206 amino acids, respectively. An almostidentical superimposition (2.49 Å for 208 amino acids) wasobtained for the eukaryotic trans-sialidase from leech (45) (Pro-tein Data Bank code 2SLI). The comparison of AAL with bac-terial sialidase is shown in Fig. 5. Structural sequence align-ment only confirms the conservation of hydrophobic aminoacids that line the junction zone of the blades. No clear se-quence similarities could be detected, and of the nine catalytic

TABLE IVEquilibrium dissociation and association constants for the interaction between fucose and AAL measured by SPR experiments

Ligand KD KA

�M 104M

�1

L-Fucose 24.1 � 1.7 4.15�-L-Fuc(13 2)-D-Gal 11.7 � 1.0 8.55�-L-Fuc(1 3 2)�-D-Gal(1 4)-D-Glc 102.9 � 4.3 0.97Blood group A trisaccharide: �-L-Fuc-(1 3 2)-[�-D-GalNAc-(1 3 3)]-D-Gal 20.5 � 1.4 4.88Blood group B trisaccharide: �-L-Fuc-(1 3 2)-[�-D-Gal-(133)]-D-Gal 23.4 � 2.3 4.27Blood group H type II trisaccharide: �-L-Fuc-(1 3 2)-�-D-Gal-(1 34)-D-GlcNAc 59.0 � 4.7 1.69Lewis a trisaccharide: �-L-Fuc-(1 34)[�-D-Gal-(1 33)]-D-GlcNAc 89.5 � 6.3 1.12Lewis X trisaccharide: �-L-Fuc-(1 33)[�-D-Gal-(1 34)]-D-GlcNAc 103.9 � 4.4 0.97p-Nitrophenyl-�-L-fucoside 43.1 � 3.1 2.32p-Nitrophenyl-�-L-fucoside 93.0 � 8.3 1.08

FIG. 4. Sensorgram of the interac-tion of increasing amounts of �-L-Fuc-(132)-�-D-Gal to the immobi-lized AAL at 25 °C. The sugarconcentration ranges from 0.39 �M (lowestcurve) to 200 �M (top curve). Each injec-tion was of 360-s duration at a flow rate of5 �l/ml. Inset, Scatchard plot of thesensorgram.

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amino acids responsible for cleaving the sialic acid from glyco-conjugates, only two are conserved in the AAL sequence. At thepresent stage, it is difficult to conclude whether a phylogeneticlink exists between sialidases and AAL.

Most of the different propeller superfamilies have evolved inrigidifying the structure by a “Velcro” closure that brings to-gether the C- and N-terminal moiety as part of the same blade(i.e. the C-terminal peptide in the position of strand 1 of thefirst blade (or alternatively the N-terminal peptide as strand 4of the last blade)). AAL does bring the two extremities of thechain together in antiparallel association but in a differentdomain, independent from the blade, looking therefore morelike a “zipper” than Velcro.

AAL Repeats in Pathogenic Microorganisms—AAL-like lec-tins have been purified using fucose affinity columns from thefruiting bodies of other mushrooms. The sequence identity of 12amino acids of 20 has been demonstrated between the N-ter-minal sequence of a lectin from another ascomycete mushroom,Melastiza chateri, and AAL (46).

Ascomycetes fungi also include species that can be patho-genic to plants or animals. Two proteins presenting six se-quence repeats highly similar to the ones of AAL have beenrecently identified in two Aspergillus species: Aspergillusoryzae (47), a plant pathogen used for fermentation of rice insake production, and A. fumigatus,3 a saprophytic fungal thatcan turn into a dangerous human pathogen in hospital envi-ronments. These two lectin sequences have 82% identity anddisplay about 30% identity with the AAL sequence. The 310-amino acid protein from A. oryzae has hemagglutinin activitythat is inhibited by L-fucose, whereas D-mannose and neura-minic acids are only weak inhibitors (47). The 314-amino acidprotein from A. fumigatus is described as fucose-lectin in thesequence deposition but also seems to correspond to a recentlydescribed 32-kDa protein specific for sialic acid (48). This dis-crepancy is hypothesized to result from differences in strains orculture conditions.

The AAL repeat has also been identified in a different orga-nism, the plant pathogenic bacterium Ralstonia solanacearum

(49), which, like Aspergillus and Aleuria, is a soil inhabitant.The R. solanacearum lectin shares the same specificity profileas AAL; it is specific for L-fucose and interacts with all fucose-bearing blood group oligosaccharides. Interestingly, the 91-amino acid sequence only contains two of the characteristicrepeats. The alignment displayed in Fig. 6 demonstrates thatthe amino acids needed to establish the five conserved hydro-gen bonds and the two strong hydrophobic contacts are con-served in the two repeats of R. solanacearum lectin and in fourrepeats of the Aspergillus lectins. In two binding sites of A.fumigatus lectin and A. oryzae lectin, the Trp of the last�-strand that is hydrogen-bonded to fucose is not conserved,and Glu is replaced by Gln. It can be predicted that the affinityfor fucose would be somewhat decreased in these two sites.

Interestingly, from the high similarity between AAL and R.solanacearum lectin repeats, it could be predicted that R. so-lanacearum lectin associates as a trimer, thus reforming asix-bladed �-propeller. No such structure has yet been ob-served. In such a case, this bacterial protein could be an exam-ple of a “primitive” propeller, since it is currently hypothesized

3 Ishimaru, T., Kubai, S., Bernard, E. M., Tamada, S., Tong, W.,Soteropuolos, P., Perlin, D. S., and Armstrong, D., SWISS-PROT, dep-osition Q8NJT4.

FIG. 5. Orthographic representation of different �-propellerswith ligand represented as sticks. A, AAL-fucose complex. B, S.thyphimurium sialidase-sialic acid derivative complex (Protein DataBank code 2SIM). C, tachylectin-2-GlcNAc complex (Protein Data Bankcode 1TL2).

FIG. 6. Alignment of amino acid sequence repeats from AAL, N-terminal sequence of M. chateri lectin (MCL), A. fumigatus lectin(AFL), A. oryzae lectin (AOL), and R. solanacearum lectin (RSL). Amino acids that are conserved in fucose binding sites and could thereforebe interacting with the ligand are displayed on black and gray background for establishing hydrogen bonds or hydrophobic contacts, respectively.

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that the existing �-propellers have been formed by modularduplication of a four-stranded sheet motif (31).

Acknowledgments—We thank the ESRF, Grenoble, for access to syn-chrotron data collection facilities. H. Lortat-Jacob (IBS, Grenoble) isacknowledged for access and help with the use of Biacore. We thankProf. N. Gilboa-Garber (Bar-Ilan University, Israel) for bringing ourattention to AAL and for helpful scientific discussion and correction ofthe manuscript.

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Anne ImbertyJean-Frederic Sanchez, Catherine Gautier and Michaela Wimmerova, Edward Mitchell,  LECTINMODE FOR ALEURIA AURANTIAAND NOVEL FUCOSE RECOGNITION

-PROPELLER FOLDβSIX-BLADED Crystal Structure of Fungal Lectin:Glycobiology and Extracellular Matrices:

doi: 10.1074/jbc.M302642200 originally published online May 5, 20032003, 278:27059-27067.J. Biol. Chem. 

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