- Email: [email protected]
Actin molecular structure and function
Actin
molecular
structure Emil
University
and function
Reisler
of California,
Los Angeles,
California,
USA
The understanding of actin structure and function has been improved’ by comparing the atomic structure of C-actin, the model of the F-actin structure, and the properties of actin mutants. Several aspects of actin structure have been tested and good progress has been made in mapping its myosin-binding sites. The dynamic properties of actin and genetic evaluation of its cellular function are attracting increasing attention.
Current
Opinion
in Cell Biology 1993, 5:41-47
Introduction
binant actin in Escherichia coli [9] and in the indirect flight muscle of Drosophila [lo] resulted in the recovery of only small amounts of functional actin. However, milligram quantities of actin can be readily obtained from yeast [11*,12**,13**,14*] and Dictyostelium [IS**] actin mutants. Yeast actin is gaining recognition as a valid model for conventional actin. Although it does not activate the myosin ATPase as well as rabbit a-skeletal actin [ 11.1, the in vitro motility of yeast actin [ lI*] and its polymerization and DNase I binding properties are reasonably similar [ 14.1. In addition to this, because yeast only has a single essential gene for actin [ 16,171, it is well posed for genetic dissection of the role of actin and actin-binding proteins in the cell life cycle [18,19]. The recent discovery of the ACT2 gene in yeast, which codes for a protein with only about 45% sequence identity to yeast actin [ 20=,21=], may shed new light on the diversity of the actin protein family.
It is remarkable that a research topic still attracts con tinuous and growing interest 50 years after its birth. The increasing hold of actin on the imagination of biochemists as well as structural and cell biologists is rooted in its central role in muscle contraction, cell motility, cytoskeletal dynamics and other cellular functions. Much of the present excitement in actin research stems from recent breakthroughs. A major development has been the solution of the atomic structure of the G-actin molecule in complex with DNase I [ l]. Many properties of actin, such as the binding of numerous proteins to its aminoterminal segment, the inhibition of its polymerization by DNase I and chemical modifications, the exchange of bound nucleotide, and the sensitivity of Cys374 probes to its polymerization, are logically explained by considering the atomic structure of this protein [ 11. The combination of G-actin atomic structure and fiber diffraction patterns obtained from oriented actin lilaments allowed Holmes et nl. [2] to construct a model of F-actin structure to about 10 A resolution. This model agreed well with the three-dimensional reconstruction of frozen-hydrated [3] and negatively stained [4] actin iilaments. Cumulatively, the F-actin models provide a framework and a set of constraints for mapping the binding sites for actin-binding proteins. These models also reveal important features of myosin subfragment 1 (S-l) and tropomyosin binding to actin. The atomic structure of G-actin and the main features of the models for F-actin structure are discussed in several excellent reviews [5,6°,7**,8**] and the reader is referred to them for further details. Another standing of yeast pression
important development in the quest for underthe cellular functions of actin is the emergence and Dic@xtelium as attractive systems for exof actin mutants. Earlier expression of recom-
The atomic structure of G-actin, which has a prominent and deep nucleotide cleft, the local unraveling of actin filaments into two strands [ 691, and the loss of actin’s in r&-o motility after chemical modification [ 22,231, suggest that dynamic changes in actin may play an important role in the contractile process. This view was originally championed by Oosawa [24] and more recently adopted by others [ 250,261. With the advent of actin mutants it may be expected that the dynamic properties of actin will be closely scrutinized and actin’s role in the generation of force and motion will be put to the test. One example of such a study is the saturation transfer electron paramagnetic resonance CEPR) experiment using actin spin-labeled at Cys374 [27*]. Use of the Cys374 probe has showed that the interaction ‘of S-l, actin and ATP does not affect the microsecond rotational motions of actin. Engineering of cysteine residues in actin mutants for labeling with probes as well as spectroscopic [28*] and molecular dynamics methods will be needed to examine the dynamic motions in actin.
Abbreviations KM--Michaelis
acto-S-l-a complex of actin with S-l; constant for acto-S-l ATPase; S-1-myosin Tn-I-troponin I; V,,maximum
@ Current
FRET-fluorescence resonance energy transfer; subfragment 1; S-l (All-S-1 isozyme containing velocity of ATP hydrolysis by acto-S-l.
Biology
Ltd ISSN 0955-0674
Al
light
chain;
41
42
Cytoplasm
and cell motility
Actin structure
The solution of G-actin structure in the complex with DNase I [l] rests on the assumption that DNase I does not significantly alter the conformation of G-actin. While this assumption and other aspects of G-actin structure are supported by biochemical data [ll, ultimately, the solved structure will be tested by the forthcoming structures of G-actin in complex with other proteins, bound at other locations on actin. Thus, the recent crystallization of actin in complex with segment 1 of gelsolin [29”] is particularly important. The crystals diffract to beyond 2.5A and should provide critical and comparative information on G-actin structure. The solution of G-actin structure in complex with profilin will be equally important because of the perceived relationship of actin ribbons in actin-proiilin crystals to F-actin [30]. The main DNase I binding loop on actin, between residues 38 and 52 on subdomain 2, and perhaps even the nucleotide cleft which DNase 1 bridges, may be perturbed by the binding of DNase I. Such changes, if any, have little bearing on close-by S-l binding sites on subdomain 1. As shown recently [31-l, ternary complexes of the S-l isozyme containing the A2 light chain, S-1&2), DNase I and G-actin can be formed, in which DNase I activity is inhibited to a normal extent by actin. Importantly, also, the binding constant of S-l(A2) for G-actin is changed very little, if at all, by DNase I. Similar con elusions were also reached by using chemically modified actin [32]. Agreement between the solution conformation of G-actin and its crystal structure is also indicated by fluorescence resonance energy transfer (FRET) mapping of distances on G-actin between probes located at CyslO, ~ysbl, Tyr69, Cys374, the nucleotide site and the metal site [28*]. In most cases, the FEET-determined distances are in good agreement with those calculated from the crystal structure of actin, with the exception of measurements dependent on the labeling of CyslO. However, because the modification of CyslO requires mild chaotropic conditions, the validity of such measurements is questionable. It is interesting that a similar ‘opening’ of CyslO to labeling appears to be achieved upon ATP exchange for ADP [ 331. The difference between the ATP and ADP states of actin is of intrinsic interest in the context of actin polymerization [34] and the stability of its filaments, although it is not revealed in its atomic structure [ 6=]. Another aspect of actin structure that merits attention involves the carboxyl-terminal residues Lys373, Cys374 and Phe375, which are removed proteolytically before crystal growth [l]. Tryptic removal of the last two residues appeared to increase the flexibility of filaments and promote their bundling [35*]. Because the loss of Lys374 and Phe375 caused only minor changes in the secondary structure of actin [35*], these residues could be mainly involved in inter-actin contacts in the filament [ 11. The relative impunity with which Cys374 can be modified with various probes suggests some tolerance of the iilament structure to alterations in the carboxyl-terminal residues. Yet, as shown by deletion of residues 373-375
in yeast actin [36**], the changes in this region, or perhaps in the binding of some actin-binding proteins, can be lethal to cells. Clearly, the role of the carboxyl tenninus in the structure and function of actin needs to be explored by using appropriate actin mutants.
Divalent
cations and nucleotides
The reader is referred to a comprehensive review by Estes et a/. [37*] for an up-to-date account on aihnities and rates of Ca2+ and Mg2+ binding to actin, and on the relationship of these cations to nucleotide binding, and the conformation and polymerization of actin. In the context of polymerization studies, it is worth noting, but not always recognized, that a complete exchange of Cal+ for Mg’+ is not achieved under standard experimental conditions, when Mg2+ is added to CaL+--actin, and that Ca’+ chelators should be included in the exchange process [ 37.1. A renewed interest in the role of nucleotides in the structure and function of actin filaments has been stimulated by the observation of different stabilities and mechanical properties of ATP- and ADP-containing filaments [ 381. Although these iindings are consistent with the more frequent local unraveling of ADP-F-actin than that of ATP-F-actin [4], they need to be carefully assessed. One reason for caution is the fact that a recent study has not found any difference between the rigidities of ATP- and ADP-F-actin (l Newman, K Schick, S Kusiak, K Zaner, L Selden, et al., abstract, Biop/!,aJ 1992, 61437). Also, the equilibrium constants for the incorporation of ATP- and ADPG-actin into filaments are independent from the type of nucleotide bound to contiguous actin subunits [39]. This latter result does not support, but also cannot exclude, the possibility of distinct conformations of ATPand ADP-F-actin.
Actin function
The diverse cellular functions of actin, as either a component of the cytoskeleton or a major factor in muscle contraction and motility processes, involve the interactions of actin with a multitude of actin-binding proteins. A particularly intriguing example of actin-based motility in non-muscle cells is the movement of the bacterium Listeria monocytogenes through the host cell cytoplasm. In this rapidly developing area of research, the movement of Listeria was found to depend on the formation of actin filament-rich comet tails [40]. A more recent study shows that the rate of bacterial movement is equal to the rate of comet tail growth at its proximal end, suggesting that actin polymerization drives the bacterial propulsion [41**]. Proteins involved in the regulation of the rapid polymerization of actin at the bacterial surface are now under investigation. Considerable insight into how actin-binding proteins polymerize actin may be gained from advances in the
Actin molecular
studies on the polymerization of actin by S-l. A detailed analysis of this process became feasible with the documentation of the different rates at which S-l(A1) and S-1642) isozymes polymerize actin [42-44]. The polymerization reaction was proposed to involve the formation of reasonably stable (G-actin)+ complexes [44] and the presence of transient oligomeric species with an actin : S-l ratio of 2 : 1 (C Valentin-Rant, MF Carlier, abstract, Bioplys J 1992,61:416). It remains to be verified if these intermediates are indeed on the main polymerization pathway leading to the final 1:l complex of actin : S-l in F-acto-S-l. The growing number of actin-binding proteins and the different mechanisms for cellular control of their interactions with actin make it impractical to cover all new developments in this area in a short and general review. Other reviews in this issue discuss four classes of actin-binding proteins: myosin (Titus, pp 77-U), small actin monomer-binding proteins (Nachmias, pp 56-621, capping proteins (Weeds and Maciver, pp 63-69) and caldesmon (Matsumura and Yamashiro, pp 7&76). The following sections focus on actin, and review the recent progress in mapping the binding sites for myosin and the regulatory proteins tropomyosin, troponin I and caldesmon. It may be expected that when the contact sites between these proteins are known in detail, important insights into the mechanism and regulation of motile processes will follow.
Myosin
Previous biochemical and structural work has led to the suggestion that the myosin-binding sites on actin are located primarily on subdomain 1 and may include some residues in subdomain 2 (reviewed in [l-3,8**] ). Thus, the binding interactions between S-l and actin are expected to encompass the following clusters of amino acids: the negatively charged residues l-4; Asp24, Asp25 and Arg28 on the 18-28 loop; Glu93, Arg95, Glu99 and GlulOO on the 92-103 loop; and perhaps a part of the 79-92 helix. The hydrophobic component of acto-S-l interactions may be centered around the 341-349 helix on actin [1,2]. Attempts to identify the so-called weak and strong S-l binding sites on actin have not yet produced conclusive results. It is clear that the amino-terminal acidic residues 14 do not constitute the strong binding site for S-l. These residues could be blocked with antibodies [45*], replaced with histidine, asparagine, glutamine or alanine [ 12**,13*=,15**], or deleted [ 12**] from actin without large changes in rigor acttrS-1 binding. However, charge reversal at the amino terminus of p-actin, through the substitutions Asp3+Lys and Asp4+Lys, greatly weakened rigor acto-S-l binding [13**]. This suggests that while repulsive interactions between the Lys-containing amino terminus of actin and the cluster of lysines in sequence 632442 on S-l [46,47**] considerably weaken acto-S-l binding, the mere loss of attractive
structure
and function
Reisler
electrostatic interactions between these regions can be tolerated by the act-S-1 complex. Although the charged groups on the amino terminus of actin are not vital to rigor or even weak acto-S-l binding [15**,48**], they are critical for the activation of myosin ATPase activity and in vitro motility of actin filaments [ 12**,15**,49,50*]. The catalytic importance of the aminoterminal acidic residues of actin has been demonstrated unambiguously by converting th& yeast actin amino terminus to a rabbit-like AC-Asp-Glu-Asp-Glu sequence (where AC is an acetyl group) and observing a threefold increase in the catalytic efficiency of yeast actin (Vmlx/KM> with very little, if any, change in K, [51]. The fact that acto-S-l ATPase can be inhibited without a major dissociation of S-l from actin is consistent with the kinetic mechanism of actomyosin ATPase regulation by troponin and tropomyosin [ 521. The sequence 18-28 on actin has been implicated in S-l binding by nuclear magnetic resonance [53] and immunochemical work [54]. More recent studies with antibodies that target residues 18-28 (S Adams, E Reisler, abstract, Biopky J 1992, 61:440) and Dictyostelium actin mutants (K Sutoh, personal communication) show that this segment of actin is involved in the activation of the myosin ATPase activity and is not critical to the rigor binding of S-l. Similarly, the loop 92-102 on actin appears to be involved in the actomyosin ATPase activity and the motile function of actin and to a lesser extent in actomyosin binding. This is indicated by Dictyostelitrm actin mutants (K Sutoh, personal communication) and the observation that the Glu93+Lys actin mutant in the indirect flight muscle of Drosopbih fails to produce an active force while maintaining nonnal rigor tension [55-l. Another site on actin commonly implicated in the binding of S-l, and more specifically the Al light chains on S-l, includes the acidic residues 361, 363 and 364, which are close to the carboxyl terminus of actin [ 53,561. However, unexpectedly, experiments with actin peptides 356-372 and 360-372, did not reveal any significant differences in the al%nities of S-l(A1) and S-l&Z) isozymes for these peptides [57*]. Consistent with the peptide results, mutations in the cluster of acidic carboxyl-terminal residues of Dictyostelium actin did not induce isozyme-specific changes in acto-S-l interactions (K Sutoh, personal communication). Strikingly, none of the mutagenic manipulations of actin (except for charge reversal at the amino terminus) blocked the acto-S-l binding in solution. This may result from the presence of multiple contacts between the two proteins and the consequent tolerance for the loss of any individual binding site. It may be that a large weakening of acto-S-l binding requires synergistic effects associated with the loss of at least two S-l binding sites on actin. Of course, any contributions to the overall acto-S-l binding made by sites implicated in such function through structural considerations (e.g. helix 341-349) have yet to be examined. The accelerated pace and scope of the work with actin mutants promise that rapid progress will be made in mapping the actomyosin interface.
43
44
Cytoplasm
and cell motility
Tropomyosin
and troponin
I
As discussed in a recent revi& [8**], the position of tropomyosin on actin appears to be along the interface between subdomains 3 and 4, with possible contacts around Lys215, Pro307, and the ct-helix Asp222-Ser233 on actin. It has been suggested [8.*] that in the ‘off state of acdn, tropomyosin binding is shifted to subdomains1 and 2, and overlaps with some of the S-l binding sites on these subdomains. Although carbodiimide crosslInking experiments indeed suggested the binding of fropomyosin to the amino-terminal residues on actin [ 581, neither immunochemical assays [ 541 nor the recent work with p-actin mutants [50*] supported this conclusion. Even the binding of tropomyosin to charged-shifted p-actin mutant (Asp3+Lys, Asp4+Lys) was indistinguishable from the binding to wild-type p-actin. Troponin I (Tn-I) binding sites on actin have not been explored extensively. Cross-linking [ 581 and nuclear magnetic resonance methods [59] implicated residues l-7 and 18-28 on actin in the binding of Tn-I. These are the same clusters of residues that are believed to bind S-l. The consequent overlap between myosin and Tn-I binding sites on actin suggests a direct competition between these proteins for at least a limited subset of contacts with actin. Such competition may be important for the regulation of actomyosin ATPase by troponin-tropomyosin. However, it is not clear how the binding of Tn-I to the amino-terminal segment of one out of seven actins triggers co-operative transitions in the filament and inhibits acto-S-l interactions on all actin molecules. Clearly, a complete understanding of the regulation of actomyosin interactions must await a detailed mapping of tropomyosin- and Tn-I binding sites on actin in its ‘on’ and ‘off states. Such mapping should now be feasible with the help of actin mutants.
Caldesmon The regulation of actomyosin ATPase activity in smooth muscle by caldesmon, as deduced from studies on a reconstituted protein system, probably involves competition between caldesmon and myosin heads for the binding to actin [GO]. The sites implicated in caldesmon binding include residues l-7, 18-28, and the carboxylterminal amino acids on actin [61-63]. The interaction of caldesmon with the carboxyl terminus of actin has been indicated by the formation of a disuffide bond between Cys580 on caldesmon and Cys374 on actin [64*]. However, the results of fluorescence studies with Cys374labeled actin [65*] and experiments with tryptically truncated actin 166,671 are more consistent with an indirect perturbation of the actin carboxyl terminus than with a direct binding of caldesmon to this region. Although the assignment of caldesmon-binding sites on actin is probably incomplete and awaits verification by mutagenic approaches, the available information is con-
sistent so far with the idea that caldesmon and S-I-ATP compete for the binding to actin. This competition can be explained by at least partial overlap between S-l and caldesmon binding sites on actin. However, as with troponin and tropomyosin, it is not yet clear how the overlap with caldesmon over a span of one actin molecule impacts the interaction of S-l with adjacent actin monomers in F-a&n. It is possible that the regulatory effect of caldesmon is due to a combination of competition with S-l for specific sites on actin and a general steric effect.
Conclusions The research on actin has entered an exciting stage. Impressive progress is being made toward understanding the various functions of actin in structural terms. Moreover, the arsenal of molecular biological tools is proving invaluable in determining the cellular functions of actin and actin-binding proteins. The combined assault by biochemical, structural and molecular biological approaches is leading to the mapping of binding sites on actin for actin-binding proteins. Several actin-binding motifs have been detected in seemingly unrelated proteins [8*=,68] and in some cases the actin-binding properties have been verified by experiments with synthetic peptides [69]. However, it is unlikely that the contacts between actin and the proteins that bind to it are limited to a short sequence of amino acids. According to crosslinking studies, proteins that polymerize (e.g. S-l, caldesmon, a-actinin) and depolymerize (e.g. cofilin, fragmin) actin bind to the same amino-terminal acidic residues on actin. Because of its flexibility, availability and charge. density, this region may act as a ‘docking area’ for numerous proteins. The eventual effect of the bound protein on actin could then depend on other contacts formed between them. In the case of myosin, the anticipated publication of the atomic resolution structure of S-l (I Rayment, personal communication) will greatly facilitate the mapping of the acto-S-l interface. The next step, though difficult, is the preparation and structural solution of crystals of G-actin in a complex with S-l. This requires a non-polymerizable G-actin and the understanding of the differences between the G-actin and F-actin complexes with S-l [70-721. These differences have yet to be fully explored. Structural and dynamic information on actomyosin interface will also be needed for a better understanding of the role of actin in motile processes. Recent in uitro motility measurements of the force and the sliding step size generated per myosin head (T Yanagida, A Ishijima, K Saitoh, Y Harada, abstract, J Biophys 1992, 61:140) are inconsistent with the prediction of a traditional crossbridge model for force generation in muscle. Although these results are contested [73], greater attention will have to be paid to the possible dynamic motions and structural transition in actin and their role in the contractile process [25*].
Actin molecular
Acknowledgments
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1.
KABSCH W, MANNHERZ HG, SUCKD, PAI E, HOLMESKC: Atomic Structure of the Actin: DNase I Complex. Nature 1990, 3037-44.
2.
HOMES KC, POPP D. GEB~IARDW, KABSCH W: Atomic Model of the Actin Filament. Nature 1990, 347:44-49.
3.
MILUGANRA, WHI~TAKERM, SAFERD: Molecular Structure of F-actin and Location of Surface Binding Sites. Nature 1990, 348:217-221.
4.
BREMERA, MILLONIG RC, SUTI?XLIN R, ENCEL A, POUNU) TD, AEBI U: The Structural Basis for the Intrinsic Disorder of the F-actin Filament: The Lateral Slipping Model. J Cell Biol 1991, 115689-703.
5.
HOLMES KC, KABSCI~W: Muscle Proteins: Actin. Ctrrr Opin Strucl Biol 1991, 1:270-280.
and function
Reisler
P, Ktdu.So~ R: Interference with Myosln Subfragment- Biding by Site-airected Mutagenesis of Actin. Eur J Biocbem 1992, 200:35-41. Three amino.tetminal double mutants of p-actin (Asp3+Ala, Asp4-rAla; Del 3, 4; Asp3+Lys, Asp4-+Lys) were expressed in yeast. Reversalof the amino-terminal charge in the Asp3-rLys, Asp4-+Lys mutant greatly reduced rigor actomyosin binding. The polymerization of actin and DNAse I binding were not al&ted by these mutations. 13. ..
The author is supported by gtanrs from NIH (AR22031) and NSF (MCB 9206739).
structure
ASPENSTROM
14.
NEFSKYB, BR!ZTSCHER A: Yeast Actin Is Relatively WeII Behaved. Eur J Biocbem 1992, 206~949-955. this work describes a high-yield procedure for the purification of yeast actin, free of Ca2+ effects on the Mgz+-induced polymerization. Poiymerization properties, DNase I binding and tropomyosin, but not profilin, binding of yeast actin are similar to that of rabbit actin. SUTOH K, ANLXI M, SUTOH K, TOYOSHIMA YY: Site-directed Mutations of Diciyosrelium Actin: Disruption of a Negative Charge Cluster at the N-terminus. Proc Nad Acud Sci USA 1991, 88:7711-7714. Aspartic residues in the amino-terminus of Dic@?stelium actin were replaced for histidine (Aspl+His and Aspl+His, Asp4+His) and the expressed mutants were purified by ion-exchange chromography. The mutations sharply diminished the V,,,, but not the KM of the acto-S-l ATPase and inhibited the in vitro sliding of actin mutants. This work shows the critical contribution of the amino-terminal charged residues of actin to the ATP-dependent actin-myosin interaction.
15. ..
16.
Gwwnz D, SURES I: Structure of a Split Yeast Gene: Complete Nucleotide Sequence of the ActIn Gene in Saccbammyces cerevlsiae. Proc Nat1 Acud Sci USA 1990, 77~25462550.
17.
6. BREMERA, AIZBI U: The Structure of the F-actin Filament . and the Actin Molecule. Curr Opin Cell Biol 1992, 4:20-26. A consensus on the SfNCNre of the F-actin filament based on the atomic model of G-a&n and the reconstruction of electron microscope images is presented. Evidence for a dynamic filament StiUCNre is discussed.
SHORTLED, HABERJE, BOTSTEW D: Lethal Disruption of the Yeast Actin Gene by Integrative DNA Transformation. Sci ence 1982, 217:371-373.
18.
ADAMS AEM. BOTSTEIND, DRUBINDG: Requirement of Yeast Fibrin for Actin Organization and Morphogenesis in Viva Nature 1991, 354:404-408.
7.
19.
CHOWDHURYS, SMITH KW, GUSTIN MC: Osmotic Stress and Yeast Cytoskeleton: Phenotype-specific Suppression of an Actin Mutation. J &II Biol 1992, 118:561-571.
MANNHERZHG: Crystallization of Actin in Complex with Actin Binding Proteins. J Biol ffxm 1992, 267:11661-11664. ;;rief review of the main features of the atomic StnKNre of G-actin and the StNCNre of F-actin. 8.
~&BSCH W, VANDEI(ERCKHOVE J: Structure and Function of Actin. Annu RetI Biopbys Biomol Struct 1992, 21:49-76. ycomprehensive review of the atomic SttUCNre of G-actin, the model of F-actin Stt’UCNre, and of the available information on the binding sites of myosin, tropomyosin, caldesmon, cofilin, profilin, a-actinin and gelsolin on actin. 9.
FRANKELS, SOWNR, UINWANDL: The Use of Sarkosyl in Generating Soluble Protein After Bacterial Expression. Proc Null Acad Sci USA 1991, 88:1192-1196.
10.
DRLIMMONDDR, HENNESSY ES, SPARROW JC: Stability of Mutant Actin. Biocbem J 1991, 274:301-303.
11. .
KRON SJ, DRUBINDG. BOIXT~IN D, SPUDICHJA: Yeast Actin Filaments Display ATP-dependent Sliding Movement over Surfaces Coated with Rabbit Muscle Myosin. &oc N&l Acud Sci USA 1992, 89:44-470. Actin from the yeast Sacc/xtromJ~es cerec@isiaeis established as a valid model for studies on actin structure and function. Purification of milligram quantities of yeastactin is described and its sliding in the in lUtro motility assaysis documented. 12. ..
COOK RK, BLAKEW, RUUENSTEIN PA: Removal of the Aminoterminal Acidic Residues of Yeast Actin. J Biol C%em 1992, 267:9430-9436. Two amino-terminal acidic residues of yeastactin (Asp2 and Glu4) were removed by deletion and substitution. The mutants showed strong inhibition of acto-S-l ATPase activity and did not slide in the in iWo motility assays.In a newly developed in &o assay, the mutants that were generated also impaired secretion in the yeast.This work shows that although the amino.terminal acidic residues are not essential to yeast survival, they are needed for the activation of myosin ATPase.
20.
S~HWOB E, MARTINRF’:New Yeast Actin-like Gene Required Late in the Cell Cycle. Nature 1992, 355:179-182. this study describes the AC72 gene in yeast, which codes for a 391 amino acid actin-like protein that is 47% identical to yeast actin.
21. .
LE&MILL!ZR JP, HENRY G, HELFMAN DM: Identiftcation of act2, an Essential Gene in the Fission Yeast Scbizosaccharomyces pombe that Encodes a Protein Related to Actin. Proc Natl Acad Sci USA 1992, 8980-83. The AC72 gene identified in this work codes for an a&n-like protein with conserved ATP and divalent metal-binding sites, and relatively divergent sites of actinactin and actin-myosin interactions. The gene encodes a function essential for germination of haploid spores. 22.
SCHWIIXR DH, KF~ONSJ.TOYOSHIMA‘IT, SPUDICHJA, REISLERE: Subtilisin Cleavage of Actin Inhibits in Vitro Sliding Movement of Actin Filaments Over Myosin. J Cell Bid 1990, 1I ~465470.
23.
PROCHNIIZ~ICZ E, YANAGIDAT: Inhibition of Sliding Movement of F-actin by Cross-linking Emphasizes the Role of the Actin Structure in the Mechanism of Motility. J Mol Biol 1930, 216:761-772.
24.
OOSAWA F: Macromolecular Assembly of Actin. In Muscle and Non Muscle Motility. Edited by Stracher A New York: Academic Press; 1983, 1:151-216.
25. .
SCHUTT CE, LUNDBERG U: Actin as the Generator of Tension During Muscle Contraction. Proc Nat1 Acad Sci USA 1992, 89:319323. This Study presents the interesting idea that a myosin-driven change in the length of actin filament is a key step in the generation of force in muscle.
45
46
Cytoplasm 26.
and cell motility
OP~ATKA A: The Molecular pling in Muscle and in G!um 1931, 41:237-251.
Basis Other
of Chemomechanical Biological Engines.
CouBiophys
40.
TILNE~ LG. CONNELIX PS. POR‘IXOY ation by the Bacterial Pathogen Cell Biol 1990, 111:297+2988.
DA: Actin Filament Listeriu Monocytogenes.
NucleJ
OSTAI’ EM, n-lo&v DD: Rotational Dynamics of Spin-labeled F-actin During Activation of Myosin S-l ATPase Using ’ Caged ATP. BiqSysJ 1991, 59:1235-1241. .%NGXiOn tnnsfer electron par%nagnetic resonance ex~xrimenrs using spin-labeled a’ctin show that the active interaction of S. I, actin and ATP induces rotation of myosin heads relative to actin, but does not change the microsecond rotational mobility of actin, as detected by the Cys37-i probe.
THEHIOI’ JA, MITCHISON TJ. TIWEY LG, PORTNO~ DA: The Rate of Actin-based Motility of Intracellular Lisle& Monocytogenes Equals the Rate of Actin Polymerization. Na/lr,r 1992, 357:257-260. The dynamics of actin filaments in the comet tails required for Lis/eria monocytogene motility in host cells were monitored by photoactivated fluorescence. The rate of actin polymerization in the comet tail was re. lated to the rate of bacterial movement indicating that actin polymer. ization drives this movement.
28. .
42.
CI-~\,\lIss;sal’llio tion of the 342:950-953.
-13.
CHEN T, RULER Isozymes with
4-t.
VAUXI-IN-R\IUC sin Subfragment in the Absence
27. .
MIKI M. O’DONOCHLIE SI. DOS RE~II~IOS CC: Structure of Actio Observed by Fluorescence Resonance Energy Transfer Spectroscopy. .I Alrrscfe Res Cell d+ofiI 1992, 13~132-145. A good miew of distance measurements between different sites on actin by FRET, carried out mainly by the authors. The results of the.se measurements are generally in good agreement with the clistances calculated from the atomic structure of G.actin. MANNHERZ HG, GOO~H J. WAI’ M. WEEDS AG. MCIA~IGHIIN PJ: Crystallization of the Complex of Actin with Gelsolin Segment 1. J A401 Biol 1992, 226:899-901. Preliminary communication on the crystallization of the 1: 1 complex between actin and human gelsolin segment 1. The cvstals diffmct to beyond 2.5A.
41. ..
29. ..
30.
SCHL~~ CE, LINIX~RG ing in Prolilin: Actin 1989, 209735-746.
U, Mysix Crystals
J, STRAIKS N: Molecular Packand its Implications. ./ No/ MO/
31. .
CHEN T, l-Luce~n. M. REISLER E: Myosin Structural Elements of G-actin: Effects quences 39-52 and 61-69 in Subdomain Bicdmisrry 1992, 32:2%1-2946. Pmteolytic digestion experiments and binding DNase I reveal that Sl(A2) does not bind to the G-actin but does perturb the region 61-69.
Subfragment of S-l&!) 2 of
1 and on SeG-actin.
BETTACHE N, BERTRAND R. KASSAI~ R: MaleimidobenzoylG-actin: Structural Properties and Interaction with Skeletal Myosin Subfragment1. Bim!wtnisf~y 1990, 29:9Q85-9091.
33.
DKE‘WES G, FAL~ISTICI~ Ii: A Reversible sition in Muscle Actin is Caused and Uncovers Cysteinc in Position 266:5508-5513.
34.
CARLIER M-F: Actin: Protein Structure ics. J Biol G’~ern 1991, 266:lA.
by
Conformational Nucleotide 10. .j Biol
TranExchange UJ~JTI 1991,
and
Filament
Dynam-
O’DONOGHLIE SI, MIKI M, DOS REMKXOS CG: Removing the TWO C-terminal Residues of Actin Affects the Filament Structure. AI-~/J Bifxhem BicyYtys 1992, 293:110-l 16. This study presents interesting resule showing destabilization of actin filaments due to pmteolytic removal of Cys374 and Phe375 from actin. JOHANNE~ F-J. GALLWITZ D: Site-directed Mutagenesis of the Yeast Actin Gene: A Test for Actin Function in Viuo. W4EO J 1991. 10:3951-3958. Several residues on actin previously implicated in actin-actin intenctions or the binding of actin-binding proteins were replaced to test actin function irz t&ro. Substitution of Lysl91, Lys336, Trp3j6, Lys373 and Cys374 had no effect on cell survival. Deletion of the carboxyl. terminal LyKys-Phe-COOH and a substitution of Asp1 1 were lethal. 37. .
E~TE.. JE, SEILXN IA, KINOSLW HJ, GERSI-I&KN LC: Tightly bound Divalent Cation of Actin. J ~Clt~scle Res Ceil Alofil 1992, 13~272-284. An excellent rwiew on all aspect5 of divalent cation interactions with actin.
39.
OHM T, WECNER and ADP-Actin.
S, O~T!ZR GF, kw on Actin Filament
A Random Bio&mirl~~
E: Interactions of G-Actin. RiodwmMry
Myosin 1991,
CharacterizaNafwe 1989,
Subfragment 30:45-X&552.
1
C, COhll$EA~l C. CARIJKR M.F. PAhTAlX3NI D: Myo1 Interacts with Two G-actin Molecules of ATP. J Biol Clwm 1991, 266:17872-17879.
DASG~IPTA G. RlilSlIR E: Nucleotide-induced Changes in the Interaction of Myosin Subfragment 1 with Actin: Detection by Antibodies Against the N-terminal Segment of Actin. Rio chwrisfry 1991, 30:9961-9966. Fah fragments of antibodies against residues 1-7 on aactin greatly Jecre-.tsed the binding of S-l to actin in the presence of MgADP, MgPPi and MgAMP-PNP. In the absence of nucleotides, the decrease in S-l binding to actin was relatively small and both S-l and Fab could bind to actin. These results suggest different roles for the actin amino temmi. nus in the binding of S.1 in the presence and absence of nuclrotides. 46.
CHAIISSEPIED P, MORALES MF: Modifying Preselected Sites on Proteins: The Stretch of Residues 633-642 of the Myosin Heavy Chain is Part of the Actin-binding Site. f’1.0~’ Na/l Acad Sci USA 1988, 85~7471-7475.
47. ..
YAhlAhlOTO K: Identification of the Site Important for the Actin-activated MgATPase Activity of Myosin Subfragment 1. J A401 Rio/ 1991, 217:223-233. l’roteol!lic digestions of S.1 at the 50120 kD junctions, i.e. within the 633&2 sequence, are employed in this work to show that the in. te@y of the Lys-Lys-Lys sequence in the 633442 stretch of residues is important for the acti\lltion of myosin ATPase by actin. DASGLVTA G. RIZI~K E: Acto-myosin Interactions in the Presence of ATP and the N-terminal Segment of Actin. Rio&w isfgv 1992, 31:1836-1841. Antibodies against the amino-terminal residues of actin (l-7) were shown to inhibit the actomyosin ATPdse activity hy both decreasing the binding of S-ATP to actin and inhibiting a catalytic step in ATP hydrolysis hy actomyosin. The catalytic inhibition is analogous to the results obtained in the studies with actin mu[anrs. See 112*“,lj~*]. 19.
36. ..
JANMEY PA, Hwrrr JH: Effect of ATP 34795-99.
Isolation and Head Complex.
48. ..
35. .
38.
AA:
45. .
measurements with sequence 39-52 on
32.
P, KASPIVX G-Actin-Myosin
J. STOSSLL TP, HAH’wx Stiffness. Narure 1990,
Copolymerization 1991, 26:11193-l
of ATP-Actin 1197.
DASGUPTA G, REISU:R E: Antibody Against the Amino Terminus of a-Actin Inhibits Actomyosin Interactions in the Presence of ATP. J MO/ Biol 1989, 207:833-836.
50. .
ASPENSTROM P, LINDBERG U. KARLSSON R: Site-specific Aminoterminal Mutants of Yeast-expressed P-Actin. FEES LeN 1992, 303:5’+63. Neutml and charge-shifting substitutions of p-actin at posirions 3 and 4 are shown to inhibit actomyosin ATPase strongly and. to ;I Ies%er extent, the in Mf?D sliding of actin filaments, and to have no effect on the hinding of tropomyosin to actin. 51.
COOK hanced tivity Yeast
RK, ROOT Stimulation by Addition Actin NH,
D, IMILLLR C, RlilwR E, R~‘l3r:.NsIl:IN PA of Myosin Subfragment 1 ATPase of Negatively Charged Residues to Terminus. J Rio/ &WI 1993, in press.
52.
CHALOVICH J, ATPase Activity ing the Binding 257:2432-2437.
53.
TRAYER I, TRAYER HR. nal Region of Al-Light
EISENIVXRG E: Inhibition by Troponin-Tropomyosin of Myosin to Actin.
~?INE DA: Evidence Chain of Myosin
EnActhe
of Actomyosin without BlockJ Hid Ckm 1982.
that the Interacts
N-termiDirectly
Actin molecular with the l&259-266. 54.
C-Terminal
Region
of Actin.
BfrJ
Hio&rn
1987,
MEJEAN C. ROYI% Cl, Luu,i,e JP. DI~NCOIR~’ J, BI;M;L\IIN Y, RO~~STAN C: Anti Actin Antibodies: An Immunological Ap preach to the Myosin-Actin and Tropomyosin-Actin interfaces. Hiwl~cm .I 1987. 244:57 l-577.
SI’ARROW J. Rlilin~ M, BILL E, KYK’I’ATA~ V. MOILOS J, DrRsT’ON J. HI~NNIZ? E, \VHIII: D: Functional and Ultrastructural Effects of a Missense Mutation in the Indirect Flight Muscle-specific Actin Gent of Drosophila melunogaster. J Mel Hiol 1991, 222~963-992. The suhstiturion of Glu93 for Lys in the actin gene of indirect flight muscle of Drnwphih interfered with the assembly of Z-disks and Iad to formation of myolibrillar bundles lacking all sarcomeric repeat. The mutation abolished the generation of active force but did not inhibit actomyosin binding or rigor stiffness.
55. .
56.
Srrroti K: Identification Sequence. Riochemis!y
of Myosin-binding 19X2. 2 1:365&3661.
Sites
on the
Actin
57. .
1~1we J-P. Bo~ea M, Rot’srr\N C. I~IW’,WIN Y: Localization a Myosin Subfragment-l Interaction Site on the C-terminal Part of Actin. Hiockm J 1992. 284~75-79. A peptide corresponding to ;I sequence 360-372 on actin hinds well both S-l(AI l and Sl(A2).
58.
59.
GIWWUX %, G~KXI.Y the Primary Structure 1987. 22:307-316. LEVINE
Troponin-I Riochw 60.
61.
62.
I3A.
J: Location of Actin.
MOIH AJG. PI~KI(Y with the N-terminal 1988, 172:389-397.
of the Tn-I flcrrrl /3io&b?r
SV: The Region
Binding f%o/$)s
of
Interaction Actin.
Rw
HEWUCK ME, C~LUO~ICI~ JM: Effect of Caldesmon on the ATPase Activity and the Binding of Smooth and Skeletal Myosin Subfragments to Actin. .I 13iol UJCW 1988. 263:187%1885. I~A!UIXI Muscle F-actin.
A, FAITO~W Caldesmon J Hiol UJOII
A, KASSAI~ R: Cross-linking to the NH,-terminal Region 1990, 2652231-2237.
AI&\IS S, DA~GI~I~I’A G. CI-WI.O~IW JM, chemical Evidence for the Binding the NHz-terminal Segment of Actin. 265:19652-19657.
63.
LE\INE BA. Mom AJG. A~II~~ARI~. MOHNI:T E, PATC~IELL VB, PEHHY SV: Structural Study of Gizzard Caldesmon and its Interaction with Actin. Eur J 13ir~c/~wr 1990, 193687496.
64. .
GRACIII++ to Actin.
1’. JANCSO A: Disulfide Cross-linking J Rio1 UJCW 1991, 266:20305-20310.
of Caldesmon
47
CROSI~IE R, AD~hts S, CHAIXXXI-I JM, REISLIZR E: The Interaction of Caldesmon with the COOH Terminus of Actin. J Biol cbem 1991, 266:20001-20006. Fluorescence experiments with Cys374.labeled actin and measurements of caldesmon binding to labeled and tryptically truncated actin show an indirect interaction between caldesmon and the carboxyl terminus of actin. 66.
CROSI~I~ Caldesmon of Actin.
CHALOVICI-I JM, RIZEH E: Interaction and Myosin Subfragment I with the C-terminus Biwhetn Bi@vs Res Gxnmun 1992, 184:23%245.
of
67.
MXIICH R. KOTA~(OWSKI J, DABROWSKA R: The Importance C-terminal Amino Acid Residues of Actin to the Inhibition of Actomyosin ATPase Activity by Caldesmon and Troponin I. i%f% Let1 1992, 297~237-240.
of
6U.
VANCOLII’ERNOIJE K, VANDI-:KEHCKHO\‘E J. Bil~a The Interfaces of Actin and Acunthamoeba Rio/ Chm 1991, 266:15427-15431.
RH,
MR, KORN Actobindin.
ED: /
69.
YONI:.TJ\W’, N, NISHI~A E, IIDA K, KLI~MGAI H, YAHARA I, SAKAI 1-I: A Short Sequence Responsible for Both Phosphoinositide Binding and Actin Binding Activities of Colilin. J Biol Uxm 1991. 266:10485-10489.
70.
Ctt~tww~en P, Kxmz~~ fragment 1 Interaction Cbem 1989. 26420752-20759.
71.
DA5GUlTA G. Wtimi J, CHELING P, REISIXR E: Interactions tween G-actin and Myosin Subfragment-1: Immunochemical Probing of the NH2-terminal Segment on Actin. Biodwmisfy 1990, 298503-8508.
72.
Cohieutl C, G-actin and Cross-linking.
of Smooth of Skeletal
RUSLIS E: Immunoof Caldesmon to J Rid C/JCWI 1990,
Reisler
6% .
IO
of J
and function
The proximity of Cys580 on caldesmon to Cys374 on actin is demonstrated by fomlation of a disulfde bond between these residues. A protocol for specific modifications of Lys580 and Cysl53 on caldesmon is detailed. Although the proposed method is laborious, it should enable specific labeling of a single cysteine on caldesmon.
of
Site in Iflfrzfi
structure
73.
Dimv Myosin J Biol
D.
A: Change in the Actin-Myosin During Actin Polymerization.
CARLIER Subfragment&em 1992,
M-F:
Interaction Probed 267:14038-14046.
by
SubJ Rio/
Be-
Between Covalent
ULXDA TQP. WAKH~CK HIM. KKON SJ, SI’LIDICH JA: Quantized Velocities at Low Myosin Densities in an in Vitro Motility Assay. Nufure 1991, 352:307-311.
E Reisler. Department ular Biology Institute, 90024-1570, USA
of Chemistry and Biochemistry, and Universky of California, Lt>s Angeles,
the MolecCalifornia
molecular
structure Emil
University
and function
Reisler
of California,
Los Angeles,
California,
USA
The understanding of actin structure and function has been improved’ by comparing the atomic structure of C-actin, the model of the F-actin structure, and the properties of actin mutants. Several aspects of actin structure have been tested and good progress has been made in mapping its myosin-binding sites. The dynamic properties of actin and genetic evaluation of its cellular function are attracting increasing attention.
Current
Opinion
in Cell Biology 1993, 5:41-47
Introduction
binant actin in Escherichia coli [9] and in the indirect flight muscle of Drosophila [lo] resulted in the recovery of only small amounts of functional actin. However, milligram quantities of actin can be readily obtained from yeast [11*,12**,13**,14*] and Dictyostelium [IS**] actin mutants. Yeast actin is gaining recognition as a valid model for conventional actin. Although it does not activate the myosin ATPase as well as rabbit a-skeletal actin [ 11.1, the in vitro motility of yeast actin [ lI*] and its polymerization and DNase I binding properties are reasonably similar [ 14.1. In addition to this, because yeast only has a single essential gene for actin [ 16,171, it is well posed for genetic dissection of the role of actin and actin-binding proteins in the cell life cycle [18,19]. The recent discovery of the ACT2 gene in yeast, which codes for a protein with only about 45% sequence identity to yeast actin [ 20=,21=], may shed new light on the diversity of the actin protein family.
It is remarkable that a research topic still attracts con tinuous and growing interest 50 years after its birth. The increasing hold of actin on the imagination of biochemists as well as structural and cell biologists is rooted in its central role in muscle contraction, cell motility, cytoskeletal dynamics and other cellular functions. Much of the present excitement in actin research stems from recent breakthroughs. A major development has been the solution of the atomic structure of the G-actin molecule in complex with DNase I [ l]. Many properties of actin, such as the binding of numerous proteins to its aminoterminal segment, the inhibition of its polymerization by DNase I and chemical modifications, the exchange of bound nucleotide, and the sensitivity of Cys374 probes to its polymerization, are logically explained by considering the atomic structure of this protein [ 11. The combination of G-actin atomic structure and fiber diffraction patterns obtained from oriented actin lilaments allowed Holmes et nl. [2] to construct a model of F-actin structure to about 10 A resolution. This model agreed well with the three-dimensional reconstruction of frozen-hydrated [3] and negatively stained [4] actin iilaments. Cumulatively, the F-actin models provide a framework and a set of constraints for mapping the binding sites for actin-binding proteins. These models also reveal important features of myosin subfragment 1 (S-l) and tropomyosin binding to actin. The atomic structure of G-actin and the main features of the models for F-actin structure are discussed in several excellent reviews [5,6°,7**,8**] and the reader is referred to them for further details. Another standing of yeast pression
important development in the quest for underthe cellular functions of actin is the emergence and Dic@xtelium as attractive systems for exof actin mutants. Earlier expression of recom-
The atomic structure of G-actin, which has a prominent and deep nucleotide cleft, the local unraveling of actin filaments into two strands [ 691, and the loss of actin’s in r&-o motility after chemical modification [ 22,231, suggest that dynamic changes in actin may play an important role in the contractile process. This view was originally championed by Oosawa [24] and more recently adopted by others [ 250,261. With the advent of actin mutants it may be expected that the dynamic properties of actin will be closely scrutinized and actin’s role in the generation of force and motion will be put to the test. One example of such a study is the saturation transfer electron paramagnetic resonance CEPR) experiment using actin spin-labeled at Cys374 [27*]. Use of the Cys374 probe has showed that the interaction ‘of S-l, actin and ATP does not affect the microsecond rotational motions of actin. Engineering of cysteine residues in actin mutants for labeling with probes as well as spectroscopic [28*] and molecular dynamics methods will be needed to examine the dynamic motions in actin.
Abbreviations KM--Michaelis
acto-S-l-a complex of actin with S-l; constant for acto-S-l ATPase; S-1-myosin Tn-I-troponin I; V,,maximum
@ Current
FRET-fluorescence resonance energy transfer; subfragment 1; S-l (All-S-1 isozyme containing velocity of ATP hydrolysis by acto-S-l.
Biology
Ltd ISSN 0955-0674
Al
light
chain;
41
42
Cytoplasm
and cell motility
Actin structure
The solution of G-actin structure in the complex with DNase I [l] rests on the assumption that DNase I does not significantly alter the conformation of G-actin. While this assumption and other aspects of G-actin structure are supported by biochemical data [ll, ultimately, the solved structure will be tested by the forthcoming structures of G-actin in complex with other proteins, bound at other locations on actin. Thus, the recent crystallization of actin in complex with segment 1 of gelsolin [29”] is particularly important. The crystals diffract to beyond 2.5A and should provide critical and comparative information on G-actin structure. The solution of G-actin structure in complex with profilin will be equally important because of the perceived relationship of actin ribbons in actin-proiilin crystals to F-actin [30]. The main DNase I binding loop on actin, between residues 38 and 52 on subdomain 2, and perhaps even the nucleotide cleft which DNase 1 bridges, may be perturbed by the binding of DNase I. Such changes, if any, have little bearing on close-by S-l binding sites on subdomain 1. As shown recently [31-l, ternary complexes of the S-l isozyme containing the A2 light chain, S-1&2), DNase I and G-actin can be formed, in which DNase I activity is inhibited to a normal extent by actin. Importantly, also, the binding constant of S-l(A2) for G-actin is changed very little, if at all, by DNase I. Similar con elusions were also reached by using chemically modified actin [32]. Agreement between the solution conformation of G-actin and its crystal structure is also indicated by fluorescence resonance energy transfer (FRET) mapping of distances on G-actin between probes located at CyslO, ~ysbl, Tyr69, Cys374, the nucleotide site and the metal site [28*]. In most cases, the FEET-determined distances are in good agreement with those calculated from the crystal structure of actin, with the exception of measurements dependent on the labeling of CyslO. However, because the modification of CyslO requires mild chaotropic conditions, the validity of such measurements is questionable. It is interesting that a similar ‘opening’ of CyslO to labeling appears to be achieved upon ATP exchange for ADP [ 331. The difference between the ATP and ADP states of actin is of intrinsic interest in the context of actin polymerization [34] and the stability of its filaments, although it is not revealed in its atomic structure [ 6=]. Another aspect of actin structure that merits attention involves the carboxyl-terminal residues Lys373, Cys374 and Phe375, which are removed proteolytically before crystal growth [l]. Tryptic removal of the last two residues appeared to increase the flexibility of filaments and promote their bundling [35*]. Because the loss of Lys374 and Phe375 caused only minor changes in the secondary structure of actin [35*], these residues could be mainly involved in inter-actin contacts in the filament [ 11. The relative impunity with which Cys374 can be modified with various probes suggests some tolerance of the iilament structure to alterations in the carboxyl-terminal residues. Yet, as shown by deletion of residues 373-375
in yeast actin [36**], the changes in this region, or perhaps in the binding of some actin-binding proteins, can be lethal to cells. Clearly, the role of the carboxyl tenninus in the structure and function of actin needs to be explored by using appropriate actin mutants.
Divalent
cations and nucleotides
The reader is referred to a comprehensive review by Estes et a/. [37*] for an up-to-date account on aihnities and rates of Ca2+ and Mg2+ binding to actin, and on the relationship of these cations to nucleotide binding, and the conformation and polymerization of actin. In the context of polymerization studies, it is worth noting, but not always recognized, that a complete exchange of Cal+ for Mg’+ is not achieved under standard experimental conditions, when Mg2+ is added to CaL+--actin, and that Ca’+ chelators should be included in the exchange process [ 37.1. A renewed interest in the role of nucleotides in the structure and function of actin filaments has been stimulated by the observation of different stabilities and mechanical properties of ATP- and ADP-containing filaments [ 381. Although these iindings are consistent with the more frequent local unraveling of ADP-F-actin than that of ATP-F-actin [4], they need to be carefully assessed. One reason for caution is the fact that a recent study has not found any difference between the rigidities of ATP- and ADP-F-actin (l Newman, K Schick, S Kusiak, K Zaner, L Selden, et al., abstract, Biop/!,aJ 1992, 61437). Also, the equilibrium constants for the incorporation of ATP- and ADPG-actin into filaments are independent from the type of nucleotide bound to contiguous actin subunits [39]. This latter result does not support, but also cannot exclude, the possibility of distinct conformations of ATPand ADP-F-actin.
Actin function
The diverse cellular functions of actin, as either a component of the cytoskeleton or a major factor in muscle contraction and motility processes, involve the interactions of actin with a multitude of actin-binding proteins. A particularly intriguing example of actin-based motility in non-muscle cells is the movement of the bacterium Listeria monocytogenes through the host cell cytoplasm. In this rapidly developing area of research, the movement of Listeria was found to depend on the formation of actin filament-rich comet tails [40]. A more recent study shows that the rate of bacterial movement is equal to the rate of comet tail growth at its proximal end, suggesting that actin polymerization drives the bacterial propulsion [41**]. Proteins involved in the regulation of the rapid polymerization of actin at the bacterial surface are now under investigation. Considerable insight into how actin-binding proteins polymerize actin may be gained from advances in the
Actin molecular
studies on the polymerization of actin by S-l. A detailed analysis of this process became feasible with the documentation of the different rates at which S-l(A1) and S-1642) isozymes polymerize actin [42-44]. The polymerization reaction was proposed to involve the formation of reasonably stable (G-actin)+ complexes [44] and the presence of transient oligomeric species with an actin : S-l ratio of 2 : 1 (C Valentin-Rant, MF Carlier, abstract, Bioplys J 1992,61:416). It remains to be verified if these intermediates are indeed on the main polymerization pathway leading to the final 1:l complex of actin : S-l in F-acto-S-l. The growing number of actin-binding proteins and the different mechanisms for cellular control of their interactions with actin make it impractical to cover all new developments in this area in a short and general review. Other reviews in this issue discuss four classes of actin-binding proteins: myosin (Titus, pp 77-U), small actin monomer-binding proteins (Nachmias, pp 56-621, capping proteins (Weeds and Maciver, pp 63-69) and caldesmon (Matsumura and Yamashiro, pp 7&76). The following sections focus on actin, and review the recent progress in mapping the binding sites for myosin and the regulatory proteins tropomyosin, troponin I and caldesmon. It may be expected that when the contact sites between these proteins are known in detail, important insights into the mechanism and regulation of motile processes will follow.
Myosin
Previous biochemical and structural work has led to the suggestion that the myosin-binding sites on actin are located primarily on subdomain 1 and may include some residues in subdomain 2 (reviewed in [l-3,8**] ). Thus, the binding interactions between S-l and actin are expected to encompass the following clusters of amino acids: the negatively charged residues l-4; Asp24, Asp25 and Arg28 on the 18-28 loop; Glu93, Arg95, Glu99 and GlulOO on the 92-103 loop; and perhaps a part of the 79-92 helix. The hydrophobic component of acto-S-l interactions may be centered around the 341-349 helix on actin [1,2]. Attempts to identify the so-called weak and strong S-l binding sites on actin have not yet produced conclusive results. It is clear that the amino-terminal acidic residues 14 do not constitute the strong binding site for S-l. These residues could be blocked with antibodies [45*], replaced with histidine, asparagine, glutamine or alanine [ 12**,13*=,15**], or deleted [ 12**] from actin without large changes in rigor acttrS-1 binding. However, charge reversal at the amino terminus of p-actin, through the substitutions Asp3+Lys and Asp4+Lys, greatly weakened rigor acto-S-l binding [13**]. This suggests that while repulsive interactions between the Lys-containing amino terminus of actin and the cluster of lysines in sequence 632442 on S-l [46,47**] considerably weaken acto-S-l binding, the mere loss of attractive
structure
and function
Reisler
electrostatic interactions between these regions can be tolerated by the act-S-1 complex. Although the charged groups on the amino terminus of actin are not vital to rigor or even weak acto-S-l binding [15**,48**], they are critical for the activation of myosin ATPase activity and in vitro motility of actin filaments [ 12**,15**,49,50*]. The catalytic importance of the aminoterminal acidic residues of actin has been demonstrated unambiguously by converting th& yeast actin amino terminus to a rabbit-like AC-Asp-Glu-Asp-Glu sequence (where AC is an acetyl group) and observing a threefold increase in the catalytic efficiency of yeast actin (Vmlx/KM> with very little, if any, change in K, [51]. The fact that acto-S-l ATPase can be inhibited without a major dissociation of S-l from actin is consistent with the kinetic mechanism of actomyosin ATPase regulation by troponin and tropomyosin [ 521. The sequence 18-28 on actin has been implicated in S-l binding by nuclear magnetic resonance [53] and immunochemical work [54]. More recent studies with antibodies that target residues 18-28 (S Adams, E Reisler, abstract, Biopky J 1992, 61:440) and Dictyostelium actin mutants (K Sutoh, personal communication) show that this segment of actin is involved in the activation of the myosin ATPase activity and is not critical to the rigor binding of S-l. Similarly, the loop 92-102 on actin appears to be involved in the actomyosin ATPase activity and the motile function of actin and to a lesser extent in actomyosin binding. This is indicated by Dictyostelitrm actin mutants (K Sutoh, personal communication) and the observation that the Glu93+Lys actin mutant in the indirect flight muscle of Drosopbih fails to produce an active force while maintaining nonnal rigor tension [55-l. Another site on actin commonly implicated in the binding of S-l, and more specifically the Al light chains on S-l, includes the acidic residues 361, 363 and 364, which are close to the carboxyl terminus of actin [ 53,561. However, unexpectedly, experiments with actin peptides 356-372 and 360-372, did not reveal any significant differences in the al%nities of S-l(A1) and S-l&Z) isozymes for these peptides [57*]. Consistent with the peptide results, mutations in the cluster of acidic carboxyl-terminal residues of Dictyostelium actin did not induce isozyme-specific changes in acto-S-l interactions (K Sutoh, personal communication). Strikingly, none of the mutagenic manipulations of actin (except for charge reversal at the amino terminus) blocked the acto-S-l binding in solution. This may result from the presence of multiple contacts between the two proteins and the consequent tolerance for the loss of any individual binding site. It may be that a large weakening of acto-S-l binding requires synergistic effects associated with the loss of at least two S-l binding sites on actin. Of course, any contributions to the overall acto-S-l binding made by sites implicated in such function through structural considerations (e.g. helix 341-349) have yet to be examined. The accelerated pace and scope of the work with actin mutants promise that rapid progress will be made in mapping the actomyosin interface.
43
44
Cytoplasm
and cell motility
Tropomyosin
and troponin
I
As discussed in a recent revi& [8**], the position of tropomyosin on actin appears to be along the interface between subdomains 3 and 4, with possible contacts around Lys215, Pro307, and the ct-helix Asp222-Ser233 on actin. It has been suggested [8.*] that in the ‘off state of acdn, tropomyosin binding is shifted to subdomains1 and 2, and overlaps with some of the S-l binding sites on these subdomains. Although carbodiimide crosslInking experiments indeed suggested the binding of fropomyosin to the amino-terminal residues on actin [ 581, neither immunochemical assays [ 541 nor the recent work with p-actin mutants [50*] supported this conclusion. Even the binding of tropomyosin to charged-shifted p-actin mutant (Asp3+Lys, Asp4+Lys) was indistinguishable from the binding to wild-type p-actin. Troponin I (Tn-I) binding sites on actin have not been explored extensively. Cross-linking [ 581 and nuclear magnetic resonance methods [59] implicated residues l-7 and 18-28 on actin in the binding of Tn-I. These are the same clusters of residues that are believed to bind S-l. The consequent overlap between myosin and Tn-I binding sites on actin suggests a direct competition between these proteins for at least a limited subset of contacts with actin. Such competition may be important for the regulation of actomyosin ATPase by troponin-tropomyosin. However, it is not clear how the binding of Tn-I to the amino-terminal segment of one out of seven actins triggers co-operative transitions in the filament and inhibits acto-S-l interactions on all actin molecules. Clearly, a complete understanding of the regulation of actomyosin interactions must await a detailed mapping of tropomyosin- and Tn-I binding sites on actin in its ‘on’ and ‘off states. Such mapping should now be feasible with the help of actin mutants.
Caldesmon The regulation of actomyosin ATPase activity in smooth muscle by caldesmon, as deduced from studies on a reconstituted protein system, probably involves competition between caldesmon and myosin heads for the binding to actin [GO]. The sites implicated in caldesmon binding include residues l-7, 18-28, and the carboxylterminal amino acids on actin [61-63]. The interaction of caldesmon with the carboxyl terminus of actin has been indicated by the formation of a disuffide bond between Cys580 on caldesmon and Cys374 on actin [64*]. However, the results of fluorescence studies with Cys374labeled actin [65*] and experiments with tryptically truncated actin 166,671 are more consistent with an indirect perturbation of the actin carboxyl terminus than with a direct binding of caldesmon to this region. Although the assignment of caldesmon-binding sites on actin is probably incomplete and awaits verification by mutagenic approaches, the available information is con-
sistent so far with the idea that caldesmon and S-I-ATP compete for the binding to actin. This competition can be explained by at least partial overlap between S-l and caldesmon binding sites on actin. However, as with troponin and tropomyosin, it is not yet clear how the overlap with caldesmon over a span of one actin molecule impacts the interaction of S-l with adjacent actin monomers in F-a&n. It is possible that the regulatory effect of caldesmon is due to a combination of competition with S-l for specific sites on actin and a general steric effect.
Conclusions The research on actin has entered an exciting stage. Impressive progress is being made toward understanding the various functions of actin in structural terms. Moreover, the arsenal of molecular biological tools is proving invaluable in determining the cellular functions of actin and actin-binding proteins. The combined assault by biochemical, structural and molecular biological approaches is leading to the mapping of binding sites on actin for actin-binding proteins. Several actin-binding motifs have been detected in seemingly unrelated proteins [8*=,68] and in some cases the actin-binding properties have been verified by experiments with synthetic peptides [69]. However, it is unlikely that the contacts between actin and the proteins that bind to it are limited to a short sequence of amino acids. According to crosslinking studies, proteins that polymerize (e.g. S-l, caldesmon, a-actinin) and depolymerize (e.g. cofilin, fragmin) actin bind to the same amino-terminal acidic residues on actin. Because of its flexibility, availability and charge. density, this region may act as a ‘docking area’ for numerous proteins. The eventual effect of the bound protein on actin could then depend on other contacts formed between them. In the case of myosin, the anticipated publication of the atomic resolution structure of S-l (I Rayment, personal communication) will greatly facilitate the mapping of the acto-S-l interface. The next step, though difficult, is the preparation and structural solution of crystals of G-actin in a complex with S-l. This requires a non-polymerizable G-actin and the understanding of the differences between the G-actin and F-actin complexes with S-l [70-721. These differences have yet to be fully explored. Structural and dynamic information on actomyosin interface will also be needed for a better understanding of the role of actin in motile processes. Recent in uitro motility measurements of the force and the sliding step size generated per myosin head (T Yanagida, A Ishijima, K Saitoh, Y Harada, abstract, J Biophys 1992, 61:140) are inconsistent with the prediction of a traditional crossbridge model for force generation in muscle. Although these results are contested [73], greater attention will have to be paid to the possible dynamic motions and structural transition in actin and their role in the contractile process [25*].
Actin molecular
Acknowledgments
References and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest 1.
KABSCH W, MANNHERZ HG, SUCKD, PAI E, HOLMESKC: Atomic Structure of the Actin: DNase I Complex. Nature 1990, 3037-44.
2.
HOMES KC, POPP D. GEB~IARDW, KABSCH W: Atomic Model of the Actin Filament. Nature 1990, 347:44-49.
3.
MILUGANRA, WHI~TAKERM, SAFERD: Molecular Structure of F-actin and Location of Surface Binding Sites. Nature 1990, 348:217-221.
4.
BREMERA, MILLONIG RC, SUTI?XLIN R, ENCEL A, POUNU) TD, AEBI U: The Structural Basis for the Intrinsic Disorder of the F-actin Filament: The Lateral Slipping Model. J Cell Biol 1991, 115689-703.
5.
HOLMES KC, KABSCI~W: Muscle Proteins: Actin. Ctrrr Opin Strucl Biol 1991, 1:270-280.
and function
Reisler
P, Ktdu.So~ R: Interference with Myosln Subfragment- Biding by Site-airected Mutagenesis of Actin. Eur J Biocbem 1992, 200:35-41. Three amino.tetminal double mutants of p-actin (Asp3+Ala, Asp4-rAla; Del 3, 4; Asp3+Lys, Asp4-+Lys) were expressed in yeast. Reversalof the amino-terminal charge in the Asp3-rLys, Asp4-+Lys mutant greatly reduced rigor actomyosin binding. The polymerization of actin and DNAse I binding were not al&ted by these mutations. 13. ..
The author is supported by gtanrs from NIH (AR22031) and NSF (MCB 9206739).
structure
ASPENSTROM
14.
NEFSKYB, BR!ZTSCHER A: Yeast Actin Is Relatively WeII Behaved. Eur J Biocbem 1992, 206~949-955. this work describes a high-yield procedure for the purification of yeast actin, free of Ca2+ effects on the Mgz+-induced polymerization. Poiymerization properties, DNase I binding and tropomyosin, but not profilin, binding of yeast actin are similar to that of rabbit actin. SUTOH K, ANLXI M, SUTOH K, TOYOSHIMA YY: Site-directed Mutations of Diciyosrelium Actin: Disruption of a Negative Charge Cluster at the N-terminus. Proc Nad Acud Sci USA 1991, 88:7711-7714. Aspartic residues in the amino-terminus of Dic@?stelium actin were replaced for histidine (Aspl+His and Aspl+His, Asp4+His) and the expressed mutants were purified by ion-exchange chromography. The mutations sharply diminished the V,,,, but not the KM of the acto-S-l ATPase and inhibited the in vitro sliding of actin mutants. This work shows the critical contribution of the amino-terminal charged residues of actin to the ATP-dependent actin-myosin interaction.
15. ..
16.
Gwwnz D, SURES I: Structure of a Split Yeast Gene: Complete Nucleotide Sequence of the ActIn Gene in Saccbammyces cerevlsiae. Proc Nat1 Acud Sci USA 1990, 77~25462550.
17.
6. BREMERA, AIZBI U: The Structure of the F-actin Filament . and the Actin Molecule. Curr Opin Cell Biol 1992, 4:20-26. A consensus on the SfNCNre of the F-actin filament based on the atomic model of G-a&n and the reconstruction of electron microscope images is presented. Evidence for a dynamic filament StiUCNre is discussed.
SHORTLED, HABERJE, BOTSTEW D: Lethal Disruption of the Yeast Actin Gene by Integrative DNA Transformation. Sci ence 1982, 217:371-373.
18.
ADAMS AEM. BOTSTEIND, DRUBINDG: Requirement of Yeast Fibrin for Actin Organization and Morphogenesis in Viva Nature 1991, 354:404-408.
7.
19.
CHOWDHURYS, SMITH KW, GUSTIN MC: Osmotic Stress and Yeast Cytoskeleton: Phenotype-specific Suppression of an Actin Mutation. J &II Biol 1992, 118:561-571.
MANNHERZHG: Crystallization of Actin in Complex with Actin Binding Proteins. J Biol ffxm 1992, 267:11661-11664. ;;rief review of the main features of the atomic StnKNre of G-actin and the StNCNre of F-actin. 8.
~&BSCH W, VANDEI(ERCKHOVE J: Structure and Function of Actin. Annu RetI Biopbys Biomol Struct 1992, 21:49-76. ycomprehensive review of the atomic SttUCNre of G-actin, the model of F-actin Stt’UCNre, and of the available information on the binding sites of myosin, tropomyosin, caldesmon, cofilin, profilin, a-actinin and gelsolin on actin. 9.
FRANKELS, SOWNR, UINWANDL: The Use of Sarkosyl in Generating Soluble Protein After Bacterial Expression. Proc Null Acad Sci USA 1991, 88:1192-1196.
10.
DRLIMMONDDR, HENNESSY ES, SPARROW JC: Stability of Mutant Actin. Biocbem J 1991, 274:301-303.
11. .
KRON SJ, DRUBINDG. BOIXT~IN D, SPUDICHJA: Yeast Actin Filaments Display ATP-dependent Sliding Movement over Surfaces Coated with Rabbit Muscle Myosin. &oc N&l Acud Sci USA 1992, 89:44-470. Actin from the yeast Sacc/xtromJ~es cerec@isiaeis established as a valid model for studies on actin structure and function. Purification of milligram quantities of yeastactin is described and its sliding in the in lUtro motility assaysis documented. 12. ..
COOK RK, BLAKEW, RUUENSTEIN PA: Removal of the Aminoterminal Acidic Residues of Yeast Actin. J Biol C%em 1992, 267:9430-9436. Two amino-terminal acidic residues of yeastactin (Asp2 and Glu4) were removed by deletion and substitution. The mutants showed strong inhibition of acto-S-l ATPase activity and did not slide in the in iWo motility assays.In a newly developed in &o assay, the mutants that were generated also impaired secretion in the yeast.This work shows that although the amino.terminal acidic residues are not essential to yeast survival, they are needed for the activation of myosin ATPase.
20.
S~HWOB E, MARTINRF’:New Yeast Actin-like Gene Required Late in the Cell Cycle. Nature 1992, 355:179-182. this study describes the AC72 gene in yeast, which codes for a 391 amino acid actin-like protein that is 47% identical to yeast actin.
21. .
LE&MILL!ZR JP, HENRY G, HELFMAN DM: Identiftcation of act2, an Essential Gene in the Fission Yeast Scbizosaccharomyces pombe that Encodes a Protein Related to Actin. Proc Natl Acad Sci USA 1992, 8980-83. The AC72 gene identified in this work codes for an a&n-like protein with conserved ATP and divalent metal-binding sites, and relatively divergent sites of actinactin and actin-myosin interactions. The gene encodes a function essential for germination of haploid spores. 22.
SCHWIIXR DH, KF~ONSJ.TOYOSHIMA‘IT, SPUDICHJA, REISLERE: Subtilisin Cleavage of Actin Inhibits in Vitro Sliding Movement of Actin Filaments Over Myosin. J Cell Bid 1990, 1I ~465470.
23.
PROCHNIIZ~ICZ E, YANAGIDAT: Inhibition of Sliding Movement of F-actin by Cross-linking Emphasizes the Role of the Actin Structure in the Mechanism of Motility. J Mol Biol 1930, 216:761-772.
24.
OOSAWA F: Macromolecular Assembly of Actin. In Muscle and Non Muscle Motility. Edited by Stracher A New York: Academic Press; 1983, 1:151-216.
25. .
SCHUTT CE, LUNDBERG U: Actin as the Generator of Tension During Muscle Contraction. Proc Nat1 Acad Sci USA 1992, 89:319323. This Study presents the interesting idea that a myosin-driven change in the length of actin filament is a key step in the generation of force in muscle.
45
46
Cytoplasm 26.
and cell motility
OP~ATKA A: The Molecular pling in Muscle and in G!um 1931, 41:237-251.
Basis Other
of Chemomechanical Biological Engines.
CouBiophys
40.
TILNE~ LG. CONNELIX PS. POR‘IXOY ation by the Bacterial Pathogen Cell Biol 1990, 111:297+2988.
DA: Actin Filament Listeriu Monocytogenes.
NucleJ
OSTAI’ EM, n-lo&v DD: Rotational Dynamics of Spin-labeled F-actin During Activation of Myosin S-l ATPase Using ’ Caged ATP. BiqSysJ 1991, 59:1235-1241. .%NGXiOn tnnsfer electron par%nagnetic resonance ex~xrimenrs using spin-labeled a’ctin show that the active interaction of S. I, actin and ATP induces rotation of myosin heads relative to actin, but does not change the microsecond rotational mobility of actin, as detected by the Cys37-i probe.
THEHIOI’ JA, MITCHISON TJ. TIWEY LG, PORTNO~ DA: The Rate of Actin-based Motility of Intracellular Lisle& Monocytogenes Equals the Rate of Actin Polymerization. Na/lr,r 1992, 357:257-260. The dynamics of actin filaments in the comet tails required for Lis/eria monocytogene motility in host cells were monitored by photoactivated fluorescence. The rate of actin polymerization in the comet tail was re. lated to the rate of bacterial movement indicating that actin polymer. ization drives this movement.
28. .
42.
CI-~\,\lIss;sal’llio tion of the 342:950-953.
-13.
CHEN T, RULER Isozymes with
4-t.
VAUXI-IN-R\IUC sin Subfragment in the Absence
27. .
MIKI M. O’DONOCHLIE SI. DOS RE~II~IOS CC: Structure of Actio Observed by Fluorescence Resonance Energy Transfer Spectroscopy. .I Alrrscfe Res Cell d+ofiI 1992, 13~132-145. A good miew of distance measurements between different sites on actin by FRET, carried out mainly by the authors. The results of the.se measurements are generally in good agreement with the clistances calculated from the atomic structure of G.actin. MANNHERZ HG, GOO~H J. WAI’ M. WEEDS AG. MCIA~IGHIIN PJ: Crystallization of the Complex of Actin with Gelsolin Segment 1. J A401 Biol 1992, 226:899-901. Preliminary communication on the crystallization of the 1: 1 complex between actin and human gelsolin segment 1. The cvstals diffmct to beyond 2.5A.
41. ..
29. ..
30.
SCHL~~ CE, LINIX~RG ing in Prolilin: Actin 1989, 209735-746.
U, Mysix Crystals
J, STRAIKS N: Molecular Packand its Implications. ./ No/ MO/
31. .
CHEN T, l-Luce~n. M. REISLER E: Myosin Structural Elements of G-actin: Effects quences 39-52 and 61-69 in Subdomain Bicdmisrry 1992, 32:2%1-2946. Pmteolytic digestion experiments and binding DNase I reveal that Sl(A2) does not bind to the G-actin but does perturb the region 61-69.
Subfragment of S-l&!) 2 of
1 and on SeG-actin.
BETTACHE N, BERTRAND R. KASSAI~ R: MaleimidobenzoylG-actin: Structural Properties and Interaction with Skeletal Myosin Subfragment1. Bim!wtnisf~y 1990, 29:9Q85-9091.
33.
DKE‘WES G, FAL~ISTICI~ Ii: A Reversible sition in Muscle Actin is Caused and Uncovers Cysteinc in Position 266:5508-5513.
34.
CARLIER M-F: Actin: Protein Structure ics. J Biol G’~ern 1991, 266:lA.
by
Conformational Nucleotide 10. .j Biol
TranExchange UJ~JTI 1991,
and
Filament
Dynam-
O’DONOGHLIE SI, MIKI M, DOS REMKXOS CG: Removing the TWO C-terminal Residues of Actin Affects the Filament Structure. AI-~/J Bifxhem BicyYtys 1992, 293:110-l 16. This study presents interesting resule showing destabilization of actin filaments due to pmteolytic removal of Cys374 and Phe375 from actin. JOHANNE~ F-J. GALLWITZ D: Site-directed Mutagenesis of the Yeast Actin Gene: A Test for Actin Function in Viuo. W4EO J 1991. 10:3951-3958. Several residues on actin previously implicated in actin-actin intenctions or the binding of actin-binding proteins were replaced to test actin function irz t&ro. Substitution of Lysl91, Lys336, Trp3j6, Lys373 and Cys374 had no effect on cell survival. Deletion of the carboxyl. terminal LyKys-Phe-COOH and a substitution of Asp1 1 were lethal. 37. .
E~TE.. JE, SEILXN IA, KINOSLW HJ, GERSI-I&KN LC: Tightly bound Divalent Cation of Actin. J ~Clt~scle Res Ceil Alofil 1992, 13~272-284. An excellent rwiew on all aspect5 of divalent cation interactions with actin.
39.
OHM T, WECNER and ADP-Actin.
S, O~T!ZR GF, kw on Actin Filament
A Random Bio&mirl~~
E: Interactions of G-Actin. RiodwmMry
Myosin 1991,
CharacterizaNafwe 1989,
Subfragment 30:45-X&552.
1
C, COhll$EA~l C. CARIJKR M.F. PAhTAlX3NI D: Myo1 Interacts with Two G-actin Molecules of ATP. J Biol Clwm 1991, 266:17872-17879.
DASG~IPTA G. RlilSlIR E: Nucleotide-induced Changes in the Interaction of Myosin Subfragment 1 with Actin: Detection by Antibodies Against the N-terminal Segment of Actin. Rio chwrisfry 1991, 30:9961-9966. Fah fragments of antibodies against residues 1-7 on aactin greatly Jecre-.tsed the binding of S-l to actin in the presence of MgADP, MgPPi and MgAMP-PNP. In the absence of nucleotides, the decrease in S-l binding to actin was relatively small and both S-l and Fab could bind to actin. These results suggest different roles for the actin amino temmi. nus in the binding of S.1 in the presence and absence of nuclrotides. 46.
CHAIISSEPIED P, MORALES MF: Modifying Preselected Sites on Proteins: The Stretch of Residues 633-642 of the Myosin Heavy Chain is Part of the Actin-binding Site. f’1.0~’ Na/l Acad Sci USA 1988, 85~7471-7475.
47. ..
YAhlAhlOTO K: Identification of the Site Important for the Actin-activated MgATPase Activity of Myosin Subfragment 1. J A401 Rio/ 1991, 217:223-233. l’roteol!lic digestions of S.1 at the 50120 kD junctions, i.e. within the 633&2 sequence, are employed in this work to show that the in. te@y of the Lys-Lys-Lys sequence in the 633442 stretch of residues is important for the acti\lltion of myosin ATPase by actin. DASGLVTA G. RIZI~K E: Acto-myosin Interactions in the Presence of ATP and the N-terminal Segment of Actin. Rio&w isfgv 1992, 31:1836-1841. Antibodies against the amino-terminal residues of actin (l-7) were shown to inhibit the actomyosin ATPdse activity hy both decreasing the binding of S-ATP to actin and inhibiting a catalytic step in ATP hydrolysis hy actomyosin. The catalytic inhibition is analogous to the results obtained in the studies with actin mu[anrs. See 112*“,lj~*]. 19.
36. ..
JANMEY PA, Hwrrr JH: Effect of ATP 34795-99.
Isolation and Head Complex.
48. ..
35. .
38.
AA:
45. .
measurements with sequence 39-52 on
32.
P, KASPIVX G-Actin-Myosin
J. STOSSLL TP, HAH’wx Stiffness. Narure 1990,
Copolymerization 1991, 26:11193-l
of ATP-Actin 1197.
DASGUPTA G, REISU:R E: Antibody Against the Amino Terminus of a-Actin Inhibits Actomyosin Interactions in the Presence of ATP. J MO/ Biol 1989, 207:833-836.
50. .
ASPENSTROM P, LINDBERG U. KARLSSON R: Site-specific Aminoterminal Mutants of Yeast-expressed P-Actin. FEES LeN 1992, 303:5’+63. Neutml and charge-shifting substitutions of p-actin at posirions 3 and 4 are shown to inhibit actomyosin ATPase strongly and. to ;I Ies%er extent, the in Mf?D sliding of actin filaments, and to have no effect on the hinding of tropomyosin to actin. 51.
COOK hanced tivity Yeast
RK, ROOT Stimulation by Addition Actin NH,
D, IMILLLR C, RlilwR E, R~‘l3r:.NsIl:IN PA of Myosin Subfragment 1 ATPase of Negatively Charged Residues to Terminus. J Rio/ &WI 1993, in press.
52.
CHALOVICH J, ATPase Activity ing the Binding 257:2432-2437.
53.
TRAYER I, TRAYER HR. nal Region of Al-Light
EISENIVXRG E: Inhibition by Troponin-Tropomyosin of Myosin to Actin.
~?INE DA: Evidence Chain of Myosin
EnActhe
of Actomyosin without BlockJ Hid Ckm 1982.
that the Interacts
N-termiDirectly
Actin molecular with the l&259-266. 54.
C-Terminal
Region
of Actin.
BfrJ
Hio&rn
1987,
MEJEAN C. ROYI% Cl, Luu,i,e JP. DI~NCOIR~’ J, BI;M;L\IIN Y, RO~~STAN C: Anti Actin Antibodies: An Immunological Ap preach to the Myosin-Actin and Tropomyosin-Actin interfaces. Hiwl~cm .I 1987. 244:57 l-577.
SI’ARROW J. Rlilin~ M, BILL E, KYK’I’ATA~ V. MOILOS J, DrRsT’ON J. HI~NNIZ? E, \VHIII: D: Functional and Ultrastructural Effects of a Missense Mutation in the Indirect Flight Muscle-specific Actin Gent of Drosophila melunogaster. J Mel Hiol 1991, 222~963-992. The suhstiturion of Glu93 for Lys in the actin gene of indirect flight muscle of Drnwphih interfered with the assembly of Z-disks and Iad to formation of myolibrillar bundles lacking all sarcomeric repeat. The mutation abolished the generation of active force but did not inhibit actomyosin binding or rigor stiffness.
55. .
56.
Srrroti K: Identification Sequence. Riochemis!y
of Myosin-binding 19X2. 2 1:365&3661.
Sites
on the
Actin
57. .
1~1we J-P. Bo~ea M, Rot’srr\N C. I~IW’,WIN Y: Localization a Myosin Subfragment-l Interaction Site on the C-terminal Part of Actin. Hiockm J 1992. 284~75-79. A peptide corresponding to ;I sequence 360-372 on actin hinds well both S-l(AI l and Sl(A2).
58.
59.
GIWWUX %, G~KXI.Y the Primary Structure 1987. 22:307-316. LEVINE
Troponin-I Riochw 60.
61.
62.
I3A.
J: Location of Actin.
MOIH AJG. PI~KI(Y with the N-terminal 1988, 172:389-397.
of the Tn-I flcrrrl /3io&b?r
SV: The Region
Binding f%o/$)s
of
Interaction Actin.
Rw
HEWUCK ME, C~LUO~ICI~ JM: Effect of Caldesmon on the ATPase Activity and the Binding of Smooth and Skeletal Myosin Subfragments to Actin. .I 13iol UJCW 1988. 263:187%1885. I~A!UIXI Muscle F-actin.
A, FAITO~W Caldesmon J Hiol UJOII
A, KASSAI~ R: Cross-linking to the NH,-terminal Region 1990, 2652231-2237.
AI&\IS S, DA~GI~I~I’A G. CI-WI.O~IW JM, chemical Evidence for the Binding the NHz-terminal Segment of Actin. 265:19652-19657.
63.
LE\INE BA. Mom AJG. A~II~~ARI~. MOHNI:T E, PATC~IELL VB, PEHHY SV: Structural Study of Gizzard Caldesmon and its Interaction with Actin. Eur J 13ir~c/~wr 1990, 193687496.
64. .
GRACIII++ to Actin.
1’. JANCSO A: Disulfide Cross-linking J Rio1 UJCW 1991, 266:20305-20310.
of Caldesmon
47
CROSI~IE R, AD~hts S, CHAIXXXI-I JM, REISLIZR E: The Interaction of Caldesmon with the COOH Terminus of Actin. J Biol cbem 1991, 266:20001-20006. Fluorescence experiments with Cys374.labeled actin and measurements of caldesmon binding to labeled and tryptically truncated actin show an indirect interaction between caldesmon and the carboxyl terminus of actin. 66.
CROSI~I~ Caldesmon of Actin.
CHALOVICI-I JM, RIZEH E: Interaction and Myosin Subfragment I with the C-terminus Biwhetn Bi@vs Res Gxnmun 1992, 184:23%245.
of
67.
MXIICH R. KOTA~(OWSKI J, DABROWSKA R: The Importance C-terminal Amino Acid Residues of Actin to the Inhibition of Actomyosin ATPase Activity by Caldesmon and Troponin I. i%f% Let1 1992, 297~237-240.
of
6U.
VANCOLII’ERNOIJE K, VANDI-:KEHCKHO\‘E J. Bil~a The Interfaces of Actin and Acunthamoeba Rio/ Chm 1991, 266:15427-15431.
RH,
MR, KORN Actobindin.
ED: /
69.
YONI:.TJ\W’, N, NISHI~A E, IIDA K, KLI~MGAI H, YAHARA I, SAKAI 1-I: A Short Sequence Responsible for Both Phosphoinositide Binding and Actin Binding Activities of Colilin. J Biol Uxm 1991. 266:10485-10489.
70.
Ctt~tww~en P, Kxmz~~ fragment 1 Interaction Cbem 1989. 26420752-20759.
71.
DA5GUlTA G. Wtimi J, CHELING P, REISIXR E: Interactions tween G-actin and Myosin Subfragment-1: Immunochemical Probing of the NH2-terminal Segment on Actin. Biodwmisfy 1990, 298503-8508.
72.
Cohieutl C, G-actin and Cross-linking.
of Smooth of Skeletal
RUSLIS E: Immunoof Caldesmon to J Rid C/JCWI 1990,
Reisler
6% .
IO
of J
and function
The proximity of Cys580 on caldesmon to Cys374 on actin is demonstrated by fomlation of a disulfde bond between these residues. A protocol for specific modifications of Lys580 and Cysl53 on caldesmon is detailed. Although the proposed method is laborious, it should enable specific labeling of a single cysteine on caldesmon.
of
Site in Iflfrzfi
structure
73.
Dimv Myosin J Biol
D.
A: Change in the Actin-Myosin During Actin Polymerization.
CARLIER Subfragment&em 1992,
M-F:
Interaction Probed 267:14038-14046.
by
SubJ Rio/
Be-
Between Covalent
ULXDA TQP. WAKH~CK HIM. KKON SJ, SI’LIDICH JA: Quantized Velocities at Low Myosin Densities in an in Vitro Motility Assay. Nufure 1991, 352:307-311.
E Reisler. Department ular Biology Institute, 90024-1570, USA
of Chemistry and Biochemistry, and Universky of California, Lt>s Angeles,
the MolecCalifornia