Proc. Natl. Acad. Sci. USAVol. 93, pp. 21–26, January 1996

Review

Protein–protein interactions in the rigor actomyosin complexRonald A. MilliganDepartment of Cell Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037

ABSTRACT Since it has not beenpossible to crystallize the actomyosincomplex, the x-ray structures of the indi-vidual proteins together with data ob-tained by fiber diffraction and electronmicroscopy have been used to build de-tailed models of filamentous actin (f-actin) and the actomyosin rigor complex.In the f-actin model, a single monomeruses 10 surface loops and two a-helices tomake sometimes complicated interactionswith its four neighbors. In the myosinmolecule, both the essential and regula-tory light chains show considerable struc-tural homology to calmodulin. Generalprinciples are evident in their mode ofattachment to the target a-helix of themyosin heavy chain. The essential lightchain also makes contacts with otherparts of the heavy chain and with theregulatory light chain. The actomyosinrigor interface is extensive, involving in-teraction of a single myosin head withregions on two adjacent actin monomers.A number of hydrophobic residues on theapposing faces of actin and myosin con-tribute to the main binding site. This site isflanked on three sides by charged myosinsurface loops that form predominantlyionic interactions with adjacent regions ofactin. Hydrogen bonding is likely to play asignificant role in actin–actin and actin–myosin interactions since many of the con-tacts involve loops. The model building ap-proach used with actomyosin is applicableto other multicomponent assemblies of bi-ological interest and is a powerful methodfor revealing molecular interactions andproviding insights into the mode of action ofthe assemblies.

Filamentous actin (f-actin) and myosinare, respectively, the track and motorcomponents that comprise one of the ma-jor systems for molecular movement in thecell. To understand how these two mole-cules accomplish mechanochemical cou-pling, it is necessary to have a detaileddescription not only of their atomic struc-tures but also of the way in which theyinteract at various stages during the workcycle. Three-dimensional crystals of the

track or of the track–motor complex arenot available, presumably because of thedifficulty in crystallizing a linear polymerof variable length. In addition, attempts tococrystallize monomeric actin and the my-osin head have been unsuccessful. Struc-tural information on actin–actin and act-in–myosin interactions cannot thereforebe obtained by x-ray crystallographicmethods directly.

To obtain this information, a combina-tion of approaches has been required.First, x-ray crystallography has providedthe atomic structures of the individualcomponents: vertebrate myosin subfrag-ment 1 (S1) and the scallop regulatory(light-chain binding) domain have beencrystallized and solved to high resolution(1, 2). The atomic structure of actin hasbeen obtained from cocrystals of actin–DNase I, profilin–actin, and gelsolin seg-ment 1–actin (3–5). Second, EM and im-age analysis of f-actin and actomyosinrigor complexes have revealed the loca-tions, packing arrangement, and geometryof interaction of the filament componentsas well as information on the dynamicnature of some interactions (6–10). Third,x-ray fiber diffraction of oriented gels off-actin was used to obtain high-resolutiondiffraction data on the filaments (11).Finally, the results obtained from all theseapproaches were combined to build, test,and refine atomic models of the filaments(11–17).

The actomyosin complex is composedof actin, the myosin heavy chain, and twomyosin light chains—the essential lightchain and the regulatory light chain. Theinteractions I will describe in this briefreview are (i) the proposed intermolecularcontacts in the actin filament, (ii) thecontacts between the myosin S1 heavychain and the light chains, and (iii) theproposed contacts at the actin–myosininterface in the rigor complex. In eachcase, I will briefly outline the results andrationale that led to the models beforefocusing on the molecular interactions andtheir possible consequences.

Actin–Actin Contacts

The atomic structure of the actin–DNaseI complex was solved 5 years ago (3).Subsequently, very similar actin structureswere obtained from crystals of actin com-plexed with profilin (4) and with gelsolin

segment 1 (5). Viewed in the standardorientation (Fig. 1, lowest actin mono-mer), the square-shaped molecule can bedivided into four subdomains: subdomain1 at bottom right (residues 1–32, 70–144,and 338–375), subdomain 2 at top right(residues 33–69), subdomain 3 at bottomleft (residues 145–180 and 270–337), andsubdomain 4 at top left (181–269) (see ref.3). The nucleotide binding site lies roughlyin the center.

An atomic model of f-actin was built tofit data obtained by x-ray fiber diffractionof oriented f-actin gels (11). The modelhas been refined with a directed mutationalgorithm and using normal modes as re-finement parameters (15, 17). The place-ment and orientation of the monomer inthe filament model is consistent with thef-actin structure seen at '30 Å by EM andthree-dimensional image analysis and withthe location of a gold cluster label (at-tached to Cys374) determined by EM dif-ference mapping (7–10). There are someuncertainties regarding the exact position-ing of the region containing the DNase Ibinding loop (residues 35–55) and theC-terminal helix (residues 368–375) (9,10, 16, 17, 19). In addition, the loop con-taining residues 264–274 was rebuilt as anantiparallel b-sheet (11, 15, 17).

Each monomer in the filament makescontact with four others: the precedingand following monomers along the samelong pitch strand (referred to as the lp 21 and lp 1 1 monomers, respectively) andthe preceding and following monomersalong the short pitch helix (sp 2 1 and sp1 1, respectively) (Fig. 1). sp 2 1 and sp1 1 lie in the second long pitch strand. Theintermolecular interactions along the longpitch strand predominantly involve sur-face loops, so hydrogen bonding may playa significant role in the interactions. Thefour contacts at each molecular interfaceeffectively bind subdomain 3 of one mono-mer to subdomains 2 and 4 of the lp 2 1monomer (Fig. 1, blue regions). The 320–328 loop lies close to the 241–247 loop inthe monomer below (lp 2 1). Both loopshave a number of hydrophobic residuesand have a complementary charge. Thesecond interaction involves two loop–helix motifs: residues 282–295 and lp 2 1loop 197–209. Again hydrophobic andThe publication costs of this article were defrayed

in part by page charge payment. This article musttherefore be hereby marked ‘‘advertisement’’ inaccordance with 18 U.S.C. §1734 solely to indicatethis fact.

Abbreviations: f-actin, filamentous actin; S1,subfragment 1.

21

charged residues are present. The thirdinteraction pairs the loops protrudingfrom b-sheet structures in subdomains 3and 2: residues 162–176 and lp 2 1 resi-dues 38–52 (part of the DNase I bindingloop). Hydrophobic residues constitutemore than half the amino acids in each ofthese loops. The final interaction involvesresidues 146 –148 (Gly-Arg-Thr) and62–64 (Arg-Gly-Ile) in lp 2 1.

Three regions of contact lie between thetwo long pitch strands (Fig. 1, green re-gions). The least extensive interaction in-volves loop 110–112 (Leu-Asn-Pro) andsp 2 1 loop 195–197 (Glu-Arg-Gly). Themost extensive interaction involves resi-dues 261–275—a loop in the actin–DNaseI structure that was rebuilt into an anti-parallel b-sheet structure during refine-ment of the filament model. The resultingfinger-like structure has a hydrophobic tip(Phe-Ile-Gly-Met) that lies between themonomers in the second long pitch strand(11, 15, 17). This interstrand contact in-volves portions of the polypeptide chainfrom three monomers: the finger from onemonomer; loop 63–65 (Gly-Ile-Leu) andloop 38–40 (Pro-Arg-His) from the sp 21 monomer; and loop 285–288 (Cys-Asp-Ile-Asp), loop 168–172 (Gly-Tyr-Ala-Leu-Pro), and loop 146–148 (Gly-Arg-Thr) from the sp 1 1 monomer. These fiveregions of the polypeptide chain cometogether to form a hydrophobic pocketinto which the hydrophobic fingertip isinserted. Although not shown in the

model, the final region of contact for whichthere is some evidence (8, 9, 17, 19) isbetween helix 223–230 and the C-terminalhelix (residues 368–375) of monomer lp 1 1.

The f-actin model shows that each actinmonomer uses 10 surface loops and twoa-helices to make a large number of in-teractions with its four neighbors. Duringfilament formation, a substantial move-ment of the C terminus is required toallow the two a-helices to interact. Inaddition, there is evidence that interac-tions involving the DNase I binding loop(residues 35–55) can change, dependingon the divalent cations present or the stateof the nucleotide bound (9, 10).

Interactions Involving the Light Chains

Myosin light chains share considerablestructural homology with calmodulin; in-deed, calmodulin serves as the lightchain(s) in a number of unconventionalmyosins (1, 2, 20–22). Schematically, thelight chains can be thought of as having alimited number of defining structural fea-tures: they have two hydrophobic pocketsconnected by a flexible linker or expan-sion joint, with divalent cation bindingsites on the outsides of the pockets.Viewed in this way, it would seem logicalthat the pockets’ structure and thereforetheir binding characteristics could bemodulated by cation binding. Further-more, the promiscuity of binding of cal-modulin in particular can be explained by

the flexibility of the linker between thepockets. Finally, the two-site attachmentwould allow for overall high-affinity bind-ing of the molecule to the target.

The chicken skeletal muscle S1 struc-ture and the structure of the regulatorydomain of scallop myosin provide twodetailed descriptions of the interactionsbetween the light and heavy chains ofmyosin (1, 2) (Fig. 2). The target for lightchain binding is an a-helix of the so-calledIQ motif, a sequence of the general formIQXXXRGXXXRXXYyW (2, 23, 24). Inthe two structures solved, the target helixis very hydrophobic and most of theseresidues make contacts with the lightchains. The critical element in the hydro-phobic interaction appears to be the pres-ence of long chain or aromatic hydropho-bic residues separated by 12 amino acids (Iand YyW in the canonical sequence) (2,21). These residues lie on opposite sides ofthe a-helix and are enclosed by the hydro-phobic pockets of the C- and N-terminaldomains of the light chains, respectively(Fig. 2 Lower). In the case of calmodulin,the expansion joint allows binding to atarget helix in which the hydrophobic res-idues are separated by only eight aminoacids (21).

There are roughly equal numbers ofpositively charged and negatively chargedresidues on the target a-helices. However,none of the negatively charged residuesseems to participate in light-chain binding,whereas '50% of the positively charged

FIG. 1. Contacts between monomers in f-actin. Shown is a stereo pair of the polypeptide backbone of four filament monomers. One of the longpitch strands is yellow; the other strand is tan. Only the unique contacts are shown. Loops making long pitch intermolecular contacts are shownin dark blue (for lp0) and light blue (for lp 2 1). Contacts between strands are shown in dark green (lp0) and light green (sp 2 1 and sp 1 1).Illustration was prepared using MOLSCRIPT (18).

22 Review: Milligan Proc. Natl. Acad. Sci. USA 93 (1996)

residues form salt bridges andyor hydro-gen bonds with the light chains. Of thecontacts between the light chains and theheavy chain, '10% are hydrogen bondsand '90% are van der Waals interactions(2). Similar figures have been reported forcalmodulin bound to a synthetic targetpeptide (21). One of the more obviousdifferences in the binding of the two lightchains is that the essential light-chain tar-get a-helix is straight, whereas that ofregulatory light chain has a .60° bendlying roughly between the two domains ofthis light chain (1, 2) (Fig. 2).

In the scallop myosin regulatory do-main, two contacts between the lightchains are required for Ca21 triggering

and regulation. Gly117 of the regulatorylight chain participates in both contacts;its main-chain nitrogen is hydrogenbonded to the main-chain carbonyl oxygenof Phe20 of the essential light chain. Itsmain-chain carbonyl oxygen is similarlylinked to the main-chain nitrogen of Arg24

in the essential light chain. These bondsbring Gly23 in the essential light chain andGly117 in the regulatory light chain intoclose contact and stabilize the specifictriggering Ca21 binding loop in the essen-tial light chain. The loop is further stabi-lized by additional interactions betweenthe light and heavy chains (Fig. 2 Lower).Thus, binding of the triggering Ca21 iscritically dependent on contacts involvingthe heavy chain and both light chains (2).

Examination of the S1 structure sug-gests that there are contacts between theessential light chain and parts of the heavychain other than the target helix (Fig. 2Upper). Essential light-chain residues90–96 form a helix just C-terminal to theexpansion joint (corresponding to the Ehelix in calmodulin) and lie in close prox-imity to a short helix formed from heavy-chain residues 720–730. In addition, thenext light-chain helix (helix F, residues103–115) is close to a helix–loop motifnear the N terminus of the heavy chain(residues 21–31). In light of recent struc-tural data on myosin–nucleotide com-plexes (25), it would seem that these con-tacts may play a critical, if not pivotal, rolein mechanochemical coupling.

FIG. 2. Light-chain–light-chain and light-chain–heavy-chain interactions in the myosin head. Myosin heavy chains are yellow. Essential andregulatory light chains are green and red, respectively. (Upper) Stereo pair of the chicken skeletal myosin S1 structure (1). Interactions betweenthe essential light chain and parts of the heavy chain other than the target a-helix are shown in light blue. (Lower) Stereo pair of the regulatorydomain of scallop myosin (2), with ball and stick representations of some side chains in the IQ motifs. Contacts between the regulatory light chain(Gly117) and the essential light chain (Phe20 and Arg24) are shown in purple. Similar contacts probably occur in the vertebrate myosins. Illustrationwas prepared using MOLSCRIPT (18).

Review: Milligan Proc. Natl. Acad. Sci. USA 93 (1996) 23

Vertebrate striated muscle essentiallight-chain isoforms are of two types, thesmaller of which is seen in the S1 struc-ture. The larger isoform has an additional41-amino acid residue extension at the Nterminus. The presence of this isoform inmuscle alters the maximum filament slid-ing velocity (e.g., see ref. 26). There isincreasing evidence that a charged regionof this N-terminal extension interacts withthe C terminus of actin (refs. 7 and 25 andreferences therein). In the S1 structure,the N terminus of the essential light chainlies roughly in the center of the light chain,so the N-terminal extension of the largerisoform must span a distance of 70–80 Å

along the underside of the S1 to reach theactin C terminus. Roughly half of theextension (in an extended conformation)is required to cover this distance, leavinga small domain of '20 amino acid resi-dues to interact with the actin C terminus.

Actin–Myosin Rigor Interactions

The model of the actomyosin rigor com-plex was built from the f-actin filamentmodel, the x-ray structure of S1, andthree-dimensional maps of the rigor acto-myosin complex obtained by cryoelectronmicroscopy and image analysis (1, 7, 11,13, 14). Three types of essential informa-

tion were obtained by electron microscopy(6, 7, 13, 14). First, three-dimensionalmaps calculated at a resolution of '30 Årevealed the overall shape of the complexand the geometry of actomyosin interac-tion. Second, difference analysis of three-dimensional maps of actomyosin contain-ing and lacking the essential light chainrevealed the location of the light chain inthe complex. Third, the first 80 aminoacids of the heavy chain were located bycomparing two three-dimensional maps,one of which was of a myosin lacking thisdomain. Although all the three-dimen-sional maps had a resolution of '30 Å, theprecision with which the light chain and

FIG. 3. Intermolecular interactions in the actomyosin rigor complex. (Upper) Two long pitch f-actin monomers interacting with the myosin headin the rigor conformation. Docking the x-ray structure with the f-actin model was carried out as described in ref. 13. (Lower) S1 has been rotatedabout a vertical axis to expose the surface that had been in contact with actin. Elements of the main binding site are shown in blue. Hydrophobicresidues are represented by blue spheres. A lysine-rich loop comprising residues 626–647—the so-called 50ky20k loop—is not present in the x-raystructure. Residues 626 and 647 are represented by green spheres as are actin residues 1–4, 24, and 25, which are most likely involved in interactionswith the loop. The so-called familial hypertrophic cardiomyopathy loop of myosin residues 404–415 and its putative contact site with actin residues332–334 are shown in purple. Elements of the putative secondary binding site, myosin residues 567–578 and actin residues 95–100, are shown inred. Illustration was prepared using MOLSCRIPT (18).

24 Review: Milligan Proc. Natl. Acad. Sci. USA 93 (1996)

the N-terminal domain could be located inthe three-dimensional maps was betterthan '5 Å. Thus, the shape of the complex,the location of specific protein components,and the location of a domain of one com-ponent provided sufficient constraints toallow the S1 atomic structure to be posi-tioned and oriented uniquely with respect tothe f-actin filament model (13, 14).

Even a relatively casual inspection ofthe resulting model shows that providingan exact description of the interactions atthe actin–myosin interface is not an easytask. The strong constraints provided bythe EM data necessitate placing the S1 ina position that results in a collision at theactin–myosin interface. It has been sug-gested that a conformational change in theS1—perhaps closure of the deep cleft inthe molecule—would alleviate these stericproblems and allow the two molecules toachieve a precise fit (13). It is not unrea-sonable to suspect that there may also besmall changes in actin that contribute tothe expected molecular complementarity.A second problem concerns a myosin sur-face loop—the so-called 50ky20k loopcomposed of residues 626–647. There isstrong evidence that this loop participatesin actomyosin interactions (27, 28). Un-fortunately, these residues are not visual-ized in the x-ray structure and thereforethey cannot be positioned with certaintyon the actin surface. A similar situationoccurs in part of another loop (Lys567–His578), which is a good candidate for anadditional actomyosin interaction (7, 13).Although these attributes of the modeleffectively rule out an exact description ofthe actomyosin rigor interface, the mostprobable general features of the interac-tion can be described.

The rigor contact between actin and themyosin head can be divided into fourdistinct regions. A large primary bindingsite on the face of actin (Fig. 3, blue) isf lanked on three sides by additional con-tacts involving myosin surface loops (13).The primary binding site is where thecollision occurs in the model. The parts ofthe myosin molecule involved in the pri-mary site are a helix–loop–helix motif(Pro529–His558) and part of an adjacenthelix (Gln647–Lys659). These myosin sec-ondary structure elements are in veryclose proximity to a helix–loop–helix nearthe C terminus of actin (Ile341–Gln354), aloop between Ala144 and Thr148 on thesame monomer and part of the DNase Ibinding loop (His40–Gly42) on an adjacentmonomer (lp 2 1) in the filament. Al-though there are a number of potentiallycomplementary ionic and polar groups, anotable feature of the binding site is thepresence of a number of hydrophobic res-idues. Sandwiched between the actin andmyosin in this region of the model areactin residues Ala144, Ile341, Ile345, Leu349,

and Phe352. They are close to residuesPro529, Met530, Ile535, Met541, Phe542, andPro543 of myosin (Fig. 3, blue spheres).While this primary binding region clearlyinvolves hydrophobic interactions, ionicinteractions and hydrogen bonding involv-ing the peptide backbone in adjacent loopsalso seem likely.

The loops flanking this main bindingsite contribute additional contacts withactin. There is considerable evidence thatthe 50ky20k loop (Tyr626–Gln647) partici-pates in actomyosin interactions (27, 28).In the S1 whose structure was solved, thisloop contains five lysines and nine glycinesand lies above the main binding site. In themodel, this loop would be close to sixnegatively charged residues on the surfaceof actin (Asp1, Glu2, Asp3, Glu4, Asp24,and Asp25) (Fig. 3, green). It is thereforeexpected that the interaction would bepredominantly ionic in nature.

Below the main binding site, the re-solved portion of another charged loop(Lys567–His578) extends toward actin and isa good candidate for the so-called second-ary binding site visualized in the EM maps(7, 13). Again, the interactions here arelikely to be ionic since there are positivelycharged residues in the loop and they seemwell positioned to interact with actin loop95–100—possibly with Glu99 and Glu100

(Fig. 3, red).The third loop (Pro404–Lys415) lies at the

front or nose of the myosin molecule andis important for normal myosin activity(29, 30). A single amino acid change herein human b cardiac myosin (equivalent toan Arg405 to Gln change in the chickensequence) is associated with familial hy-pertrophic cardiomyopathy (30). In themodel, this myosin loop lies close to actinresidues Pro332–Glu334 (Fig. 3, purple).

In summary, the myosin rigor bindingsite on f-actin is extensive and spans thejunction between two adjacent actinmonomers in the long pitch helix. Thebinding site appears to be centered onhydrophobic interactions involving helicesat the actin and myosin surfaces. Aroundthis ‘‘greasy patch’’ in the main region ofinteraction there are complementaryionic and polar groups. Myosin surfaceloops flank this main region on threesides. The loops are well placed to allowionic interactions with the surface of actin.It seems likely that hydrogen bondinginvolving the polypeptide backbone onadjacent loops contributes significantly tothe binding site.

Conclusion

The interactions between the proteincomponents of the actomyosin rigor com-plex are extensive. The x-ray structures ofa vertebrate myosin S1 and of the scallopmyosin regulatory domain provide a de-

tailed picture of the way in which the lightchains interact with the heavy chain andwith each other. The general principlesrevealed are likely to hold true for lightchain binding throughout the myosin fam-ily. A model building approach incorpo-rating data from x-ray crystallographic,fiber diffraction, and EM studies hasproved extremely successful in revealingthe probable intermolecular contacts inf-actin and in the actomyosin rigor com-plex. In f-actin the bonding pattern ap-pears to be complex and involves predom-inantly loop interactions along and be-tween the actin long pitch strands. Oneinteraction involves loops from three mol-ecules. The interaction of actin and themyosin head in the rigor complex is veryextensive, involving two actin monomersand four discrete parts of the apposing my-osin face. There is evidence for direct bind-ing of part of the large essential light-chainisoform to the C-terminal part of actin.

Construction of a high-resolution modelof a macromolecular complex by combiningmoderate resolution EM data on the intactcomplex with the x-ray crystal structures ofits individual components is a powerful ap-proach for studying large multicomponentassemblies that are not amenable to crystal-lization or current x-ray crystallographicmethods—e.g., virus–antibody and virus–receptor complexes, muscle filaments, mo-tor–microtubule complexes, ribosomes, andnuclear pore complexes. Models built in thisway reveal structural details of the interac-tions between components and provide in-sights into the mode of action of the assem-bly (13, 31).

I am grateful to my immediate colleagues forcomments on the manuscript and for help inmaking the illustrations. R.A.M. is supportedby grants from the National Institutes of Health(AR39155, GM44932, and GM52468) and is anEstablished Investigator of the American HeartAssociation.

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