Proc. Natl. Acad. Sci. USAVol. 92, pp. 10644-10647, November 1995Biochemistry

Target sequence recognition by the calmodulin superfamily:Implications from light chain binding to the regulatorydomain of scallop myosinANNE HOUDUSSE AND CAROLYN COHEN*Rosenstiel Basic Medical Research Center, Brandeis University, Waltham, MA 02254-9110

Communicated by Gregory A. Petsko, Brandeis University, Waltham, MA, August 14, 1995 (received for review June 8, 1995)

ABSTRACT Some of the rules for how members of thecalmodulin (CaM) superfamily bind to target peptides arerevealed by the crystal structure of the regulatory domain ofscallop myosin. The structure shows that the IQ motif of theheavy chain in this invertebrate myosin imposes constraintson both the positioning and conformation of the individuallobes of the light chains. In contrast, analysis of the contactresidues in the targets bound by Ca2+-CaM reveals how thestructure ofCaM accommodates a broader range of sequencesconsonant with this protein's functional diversity.

R790

The so-called IQ motif (approximately 23-25 residues), with aconsensus sequence of the form IQXXXRGXXXR (in one-letter amino acid code, where X is any amino acid), is repeatedtandemly (2-6 times) in the heavy chain (HC) of manymyosins. This motif confers on these proteins the capacity tobind myosin-associated light chains (LCs) or calmodulin(CaM) (1). The structure determination at 2.8 A resolution ofthe regulatory domain of scallop myosin (2) provided the firstdescription of the physical basis for the binding of both theregulatory (RLC) and essential (ELC) LCs to the two IQmotifs in the HC of this myosin. [More recently the structurehas been refined to 2.0 A resolution (unpublished data).] Theresults show that the lobest of the ELC and RLC adopt threedistinct conformations when bound to the HC. Two of theseconformations have been identified previously in CaM (3) andtroponin C (TnC) (4) and correspond to the conventional"open" and "closed" forms that a lobe assumes when divalentcations are bound or absent, respectively. The third state is anunusual "semi-open" conformation in which no metal is boundand is found in the C-terminal lobes of each of the two LCs.We argue here that lobe conformation depends not only ondivalent cation binding but also on the precise pattern ofresidues in the target peptide. We also describe the implica-tions for understanding target sequence recognition by CaM,which may be relevant as well for other members of the CaMsuperfamily.A key feature of the semi-open conformation is the specific

recognition of the first part of an IQ motif (IQXXXR) bylinker III in the C-terminal lobe of both LCs (Fig. 1). As aresult, the HC helix is not located deep inside the lobe as in anopen form; rather, the HC helix is gripped by LC residuessituated at the tips of the lobe. Most of these interactions arevery similar for the lobes of each of the two LCs (2). Fourconserved hydrogen bonds are formed between the backboneatoms of linker III and the glutamine and arginine side chainsof the HC IQ motif, which project from one face of the HC.The other face of the amphipathic HC helix-which includesnot only the first but also the fifth residue of the IQ motifsequence-interacts with the apolar interior of the lobe. Wewould therefore reformulate the consensus sequence of the

FIG. 1. Semi-open conformation. The ribbon diagram of theC-terminal lobe of the ELC was generated by MOLSCRIPT (5). Thisunusual conformation is induced by the specific recognition of the firstpart (IQXXIR) of the IQ motif (ball-and-stick models) in the HC helix(black). The linker (red) between domains III (yellow) and IV (green)adopts a wider turn at the end of helix G than in either the open orclosed conformations and plays a critical role in this recognition.Compared to an open conformation, the semi-open lobe has ashallower hydrophobic pocket, and, therefore, the target helix ispositioned nearer the tips of the lobe rather than deeper in the pocket.The C-terminal lobe of the RLC interacts in a very similar fashion withthe same segment of the other IQ motif in the HC and, thus, adoptsa very similar semi-open conformation (not shown).

first part of an IQ motif as IQXXIR, although the fifth residueis not always apolar. In the binding site for the ELC, the apolar"I" residues are F785 and 1789, and in the binding site for theRLC, they are 1811 and I815. Thus it appears that thesemi-open lobe conformation is stabilized by interactions withresidues on both faces of the HC helix. This highly conservedportion of the IQ motif (IQXXIR) constitutes, we believe, themost critical region of the motif, which determines both theconformation of the C-terminal lobes of the LCs and theirpositioning on the HCs.The second part of the IQ motif (GXXXR), which binds the

N-terminal lobe of the ELC, plays a relatively minor role inaffecting lobe conformation and positioning. In scallop myosinthat is regulated, this lobe has an unusual feature: although itcontains a bound (triggering) Ca2+ ion in domain I, the

Abbreviations: HC, heavy chain; LC, light chain; CaM, calmodulin;RLC, regulatory LC; ELC, essential LC; TnC, troponin C.*To whom reprint requests should be addressed.tIn a CaM-like protein, each lobe comprises two EF-hand domains.The nomenclature for the arrangement of the domains is as follows:domain I begins with helix A, followed by loop I, then helix B followedby linker I; domain II begins with helix C, loop II, then helix D, andso on.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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liganding of this ion is different from that in a typical EF hand,and this lobe displays a closed conformation similar to thatfound in the N-terminal lobe of TnC (4) (Fig. 2). Both theconformation and positioning of this lobe in the ELC ofchicken skeletal myosin (where no Ca2+ is bound) (6) aresimilar to those of scallop ELC, so that this form of the lobeis probably a general feature in myosins. As expected from itsclosed, nongripping conformation, this lobe makes relativelyfew linkages with the HC helix. The glycine residue of this partof the motif allows a close interaction of the HC with linker Iof the ELC lobe, and the last arginine forms three hydrogenbonds. Additional interactions contribute to fixing the positionof this closed lobe of ELC on the HC, including a number ofspecific contacts with its own C-terminal lobe, as well as withthe neighboring C-terminal lobe of the RLC (2).The GXXXR part of the IQ motif is not well conserved, and

in the case of the site for binding the N-terminal lobe of theRLC, it is replaced by a pattern of three hydrophobic residues,which play a role similar to that found in CaM target peptides.Correspondingly, the N-terminal lobe of the RLC (which bindsa single Mg2+ ion in a conventional way) displays the canonicalopen conformation (Fig. 3A) characteristic of Ca2+-CaM. Inthis open conformation, the apolar surface inside the lobeforms a deep pocket at the bottom of which the HC helix ispositioned by hydrophobic interactions. A key residue of theHC (W826) acts as an anchor, followed by two other apolarresidues (Y830 and V833), which project from the same faceof the helix. This pattern is very similar to that found in theC-terminal lobe of CaM binding to a segment of the M13peptide (Fig. 3B) (7), where W4 is the corresponding anchor,followed by F8 and Vl1 on the same face. The anchoringresidue often has a bulky hydrophobic side chain while the two(or so) residues following in the pattern need only be com-

patible with the apolar interior of the open lobe. Thus in theM13 target peptide interacting with the open N-terminal lobe

A

B

CaMC

FIG. 2. Closed conformation. The ribbon diagram of the N-terminal lobe of the ELC was generated by MOLSCRIPT. Despite thebinding of a Ca2+ ion in loop I, the hydrophobic surfaces of the A/Dand B/C helical pairs pack against one another to form a closed stateof the lobe similar to that found in TnC. Because of the interruptionproduced by two glycine residues, the C helix of the scallop ELC lacks1.5 turns compared with the homologous helix of TnC, and, corre-spondingly, loop II (turquoise) is unusually long. The linker (red)between domain I (yellow) and domain II (green) interacts with theGXXXR part of the IQ motif. Compared to the "gripping" of targethelices by open or semi-open lobes, the surface interactions betweenthis closed lobe and the HC are less extensive.

PeptideM13

CaMFIG. 3. Open conformations. The ribbon diagrams were generated

by MOLSCRIPT. (A) The N-terminal lobe of the RLC bound to scallopHC. (B) The C-terminal lobe of CaM bound to the M13 peptide. (C)The N-terminal lobe of CaM bound to the M13 peptide. In the openconformation, the orientations of both the A/D and B/C helical pairsare roughly orthogonal, with no interactions between the pairs, so thatthe hydrophobic surface inside the lobe forms a deep pocket thatinteracts with the HC helix. A bulky hydrophobic residue (red), whichanchors the peptide deep inside this pocket, is an essential feature ofthe recognition pattern shown here. Comparison of the N-terminallobe of CaM bound to this peptide with the N-terminal lobe of theRLC bound to the HC shows that the direction of the peptide insidethe open lobe is reversed in the two structures (see also Fig. 4).

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B

IV

N

C

11

1-'3 ELCI

FIG. 4. Relative positioning of the two lobes of CaMsuperfamily proteins. The ribbon diagrams were generated byMOLSCRIPT. The RLC (A), the ELC (B), and CaM bound tothe M13 peptide (C) are compared with the following con-

>% ventions for colors: target helix, black; domain 1, red; domainIA 11, yellow; domain III, turquoise; domain IV, blue; linker

between the two lobes, magenta; and linker III betweendomains III and IV, green. In all these complexes, the polarityof the bound protein is opposite that of the target helix. TheLC and CaM complexes differ in the positioning of thedomains of the lobes with respect to the target peptides;moving from the N- to the C-terminal end of the peptides, theinteractions involve the protein domains in the followingorder: IV, III, I, 1I for CaM; III, IV, II, I for RLC; and III,IV, I for ELC (2).

ofCaM, the anchoring residue (F17) is followed by two alanineresidues (A14 and A10) (Fig. 3C). Because of the relativelyunspecific character of this aspect of CaM-peptide recogni-tion, a variety of apolar interactions will likely be observed as

more structures are determined [see, for example, the CaM-CaM-dependent protein kinase IIa peptide complex (7)].

In contrast to the IQ motif, which positions the two domainsof each lobe with respect to the HC, the pattern ofhydrophobicresidues that binds to an open lobe allows a certain degree ofdegeneracy in this respect: the anchoring residue may be

preceded or followed by the additional hydrophobic residues.For example, considering the N to C polarity of the targethelix, the open N-terminal lobe of the RLC (Fig. 3A) binds inthe reverse orientation to that of the N-terminal lobe of CaM(2) (Fig. 3C). Additional residues that bind to the lobes inspecific ways determine their arrangement, and these are differ-ent in the two structures. In the case ofCaM target peptides, basicresidues that make linkages to the first domain of the lobe are

located about 10 residues from the anchor (Fig. 3 B and C). Theunusual sharp bend ("hook" region) in theHC helix plays this role

Table 1. Summary of conditions affecting lobe conformation

Conditions Conformation of the lobes

Protein Metal Target sequence N terminal - C terminal Basis Source

Scallop RLC +Mg2+ First part of IQ motif Open (Mg2+) Semi-open Structure Ref. 2Scallop ELC +Ca2+ Complete IQ motif Closed Semi-open Structure Ref. 2Chicken ELC -Ca2+ Complete IQ motif Closed Semi-open Structure Ref. 6TnC -Ca2+ None Closed Open (2 Ca2+) Structure Ref. 4

+Ca2+ None Open (2 Ca2+) Open (2 Ca2+) Prediction Ref. 9CaM +Ca2+ None Open (2 Ca2+) Open (2 Ca2+) Structure Ref. 3

+Ca2+ Non-IQ motif peptide Open (2 Ca2+) Open (2 Ca2+) Structure Refs. 7 and 8-Ca2+ None Closed Closed Prediction Ref. 9-Ca2+ Complete IQ motif Closed Semi-open Prediction This paper-Ca2+ First part of IQ motif Open (2 Ca2+) Semi-open Prediction This paper

A

C

C

CaM

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Proc. Natl. Acad. Sci. USA 92 (1995) 10647

A ELCsemi - open

.AI-(Ctsrm lob.)

Nl I l.G x x x R IC

clsed(Ntem. lobe)

Complete IQ motif

B RLC

emi - open(C termn. lob)

N

x(Nterm. lbbe)xC

Incomplete IQ motif

CaM

C open (Nt.nn.lob)

NC

open

(CteNnt) oe

NoleIQ motif

FIG. 5. Schematic drawing of modes of target binding. Each halflobe corresponds to a pair of helices (e.g., A/D, B/C, F/G, E/H). Thedomains to which they belong are noted in the figure to indicate thepolarity of binding. (Domains III and I are stippled.) Note that in theELC, N-terminal lobe domain I makes only surface interactions withthe target peptide. For clarity, the complete interlobe linker is notshown in CaM (C). The details of these complexes are illustrated inFig. 4.

for the N-terminal lobe of the RLC (Fig. 3A) by allowing the sidechain ofW824 to interact strongly and specificallywith the seconddomain of the lobe. As shown in Fig. 4, the hook in the HC helixplaces the two binding sites in tandem so that "40 residues of theHC helix are stabilized by the two lobes of the RLC (Fig. 4A); incontrast, the binding sites on the target helix of CaM allow bothlobes to interact (on different sides) with the same span of targetpeptide (-20 residues) (Fig. 4C). The two lobes of the ELC bindto slightly overlapping sites so that this LC spans "30 residues ofthe HC (Fig. 4B).To predict how a non-IQ motif peptide binds to the open

lobes of CaM, some relationship between the binding sites foreach lobe must be established. Sequence comparison sug-gested that two anchors would generally be separated by 12residues (7). This "rule" was subsequently modified by therecognition of flexibility in the linker between the two lobes,so that the number of intervening residues can vary consid-erably (8). In contrast, the lobes are precisely positioned onpeptides that display the complete IQ motif. To fit this12-residue rule to scallop HC, it was suggested that the IQmotif could be extended by 3 residues (2). The rule is irrele-

vant, however, when the IQ motif of a target peptide iscomplete since the hydrophobic residues that interact withsemi-open or closed lobes do not function as anchors.Our results also provide a rationale for predicting the

conformation of CaM when bound to an IQ motif, such asthose found in nonconventional myosins (1) (see Table 1 andFig. 5). No structures are yet available for these complexes, butwe note that CaM has the residues we have identified as criticalfor scallop ELC binding to HC. We would therefore expectthat when bound to a HC with a complete IQ motif, theC-terminal lobe of CaM would be in a semi-open conforma-tion, and the N-terminal lobe would be in a closed conforma-tion; and in neither case need Ca2+ be bound (A.H., M. Silver,and C.C., unpublished data). In contrast, when only theIQXXIR part of the IQ motif is present, the N-terminal lobewould adopt an open conformation, provided that an appro-priate anchoring residue (as well as bound Ca2+) is present. If,however, key residues are missing in the first part of the IQmotif, both lobes may then be in an open state. Thesesuggestions may account for the previously puzzling findingthat some CaM binding sites on nonconventional myosin HCsare preferentially occupied at low Ca2+ concentrations,whereas others require Ca2+ to be bound (10).

These considerations also suggest how the design of differ-ent CaM-like proteins is matched to a multiplicity of functionalrequirements. For example, the role of the two LCs in con-ventional myosins is to bind selectively by means of the IQmotif to stabilize the HC helix, thus forming a domain withmechanical properties required for tension development;moreover, in certain regulated myosins, the LCs are sites ofspecific Ca2+ binding or phosphorylation and act as confor-mational switches to control the catalytic activity of themolecule. In contrast, Ca2+-CaM is able to activate manydiverse enzymes, since the open lobes provide a binding sitethat allows a flexible pattern of residues in the target peptides.The fact that CaM can also bind in a variety ofways to the HCsof nonconventional myosins displays the full versatility of thisCa2+-sensing protein.We thank Jeremy Thorner for many helpful comments on the

manuscript. This work was supported by grants to C.C. from theNational Institutes of Health (AR17346 and AR41808) and theMuscular Dystrophy Association and by postdoctoral fellowships fromEuropean Molecular Biology Organization and the Human FrontierScience Program Organization to A.H.

1. Cheney, R. J. & Mooseker, M. S. (1992) Curr. Opin. Cell Bio. 4,27-35.

2. Xie, X., Harrison, D. H., Schlichting, I., Sweet, R. M., Kalabokis,V. N., Szent-Gyorgyi, A. G. & Cohen, C. (1994) Nature (London)368, 306-312.

3. Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means,A. R. & Cook, W. J. (1985) Nature (London) 315, 37-40.

4. Herzberg, 0. & James, N. M. G. (1985) Nature (London) 313,653-659.

5. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950.6. Rayment, I., Rypniewski, W. R., Schmidt-Base, K., Smith, R.,

Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesen-berg, G. & Holden, H. M. (1993) Science 261, 50-58.

7. Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B.& Bax, A. (1992) Science 256, 632-638.

8. Meador, W. E., Means, A. R. & Quiocho, F. A. (1993) Science262, 1718-1721.

9. Strydnadka, N.C.J. & James, M.N.G. (1991) Curr. Opin. Struct.Biol. 1, 905-914.

10. Bahler, M., Kroschewski, R., Stoffler, H.-E. & Behrmann, T.(1994) J. Cell Biol. 126, 375-389.

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