Actin Structure and Function: What We Still Do Not Understand

Actin is interesting. We state this not just because actin is so plentiful that it is the single most abundant protein in many eukaryotic cells. Actin is interesting not only because it is so highly conserved that between humans and chickens there have been no amino acid changes in the 375 residues present in the skeletal muscle isoform (1Sheterline P. Clayton J. Sparrow J. Protein Profile. 1995; 2: 1-103PubMed Google Scholar). We think that actin remains interesting because after more than 60 years of study (2Feuer G. Molnar F. Pettko E. Straub F.B. Acta Physiol. Hung. 1948; 1: 150-163PubMed Google Scholar) fundamental questions about actin structure and dynamics and how these determine function remain unanswered. We will not attempt to summarize what is known about actin in this very brief review. Indeed, a search of PubMed for "actin" retrieves 53,234 publications! Rather, we will focus on a few related issues involving the structure of the actin monomer and polymer and spend as much time discussing what we still do not know about actin as we spend reviewing what we do know. Actin is found in two main states: G-actin (G for globular) is the monomeric form (Fig. 1), whereas F-actin (F for filamentous) is the helical polymer (Fig. 2). The determination of the first crystal structure for G-actin was a major breakthrough (3Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1516) Google Scholar), and the concurrent determination of a crystal structure for the ATPase domain of the heat shock protein HSC-70 (4Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (819) Google Scholar) led to the immediate realization that both of these proteins share the same fold as the enzyme hexokinase (5Flaherty K.M. McKay D.B. Kabsch W. Holmes K.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5041-5045Crossref PubMed Scopus (294) Google Scholar). All three proteins contain two major domains with a nucleotide bound in the cleft between these domains. The existence of the three structures, combined with the available information that the domains in hexokinase undergo substantial rotations with respect to each other, led to the development of an algorithm to predict other proteins in this superfamily based upon the notion that all such proteins would share both a common ATP-binding fold and hinge structure between the two major domains (6Bork P. Sander C. Valencia A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7290-7294Crossref PubMed Scopus (694) Google Scholar). Subsequent structural work has revealed that this superfamily is indeed vast and includes bacterial (7van den Ent F. Amos L.A. Löwe J. Nature. 2001; 413: 39-44Crossref PubMed Scopus (604) Google Scholar, 8van den Ent F. Moller-Jensen J. Amos L.A. Gerdes K. Löwe J. EMBO J. 2002; 21: 6935-6943Crossref PubMed Scopus (203) Google Scholar) and archaeal (9Roeben A. Kofler C. Nagy I. Nickell S. Hartl F.U. Bracher A. J. Mol. Biol. 2006; 358: 145-156Crossref PubMed Scopus (39) Google Scholar, 10Hara F. Yamashiro K. Nemoto N. Ohta Y. Yokobori S. Yasunaga T. Hisanaga S. Yamagishi A. J. Bacteriol. 2007; 189: 2039-2045Crossref PubMed Scopus (23) Google Scholar) counterparts of actin as well as enzymes such as propionate kinase (11Simanshu D.K. Savithri H.S. Murthy M.R. J. Mol. Biol. 2005; 352: 876-892Crossref PubMed Scopus (25) Google Scholar) that have no common function with actin, but share a common fold and thus presumably share a common ancestral protein. We think that looking at these actin-like proteins can be informative and may tell us something about actin itself.FIGURE 2Three-dimensional reconstruction (60Orlova A. Garner E.C. Galkin V.E. Heuser J. Mullins R.D. Egelman E.H. Nat. Struct. Mol. Biol. 2007; 14: 921-926Crossref PubMed Scopus (67) Google Scholar) at ∼12-Å resolution of F-actin from cryo-EM (gray surface) with atomic models of the actin subunit (ribbon diagrams). The four actin subdomains are labeled. At the resolution shown, minimal changes are needed to fit crystal structures of G-actin into the filament reconstruction, consistent with earlier models (33Holmes K.C. Popp D. Gebhard W. Kabsch W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1306) Google Scholar). The main contacts that are seen between protomers, from subdomain 2 of one subunit to subdomain 1 of the subunit above and from subdomain 4 of one subunit to subdomain 3 above, involve structural elements of actin that are absent in the bacterial homologs MreB (55Galkin V.E. VanLoock M.S. Orlova A. Egelman E.H. Curr. Biol. 2002; 12: 570-575Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and ParM (60Orlova A. Garner E.C. Galkin V.E. Heuser J. Mullins R.D. Egelman E.H. Nat. Struct. Mol. Biol. 2007; 14: 921-926Crossref PubMed Scopus (67) Google Scholar). Subdomain 2 of actin has been observed by EM to be the most structurally labile part of the actin subunit.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Of the ∼25 actin crystal structures that have been published, only one shows a significant opening of the nucleotide-binding cleft (12Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (183) Google Scholar). There has been some controversy about whether an opening of the cleft might be associated with the change in the bound nucleotide from ATP to ADP and whether there are other conformational changes within the subunit associated with the change in the bound nucleotide. The reason for interest in this topic is the vast literature showing how actin filament dynamics are regulated by ATP hydrolysis (13Pollard T.D. Blanchoin L. Mullins R.D. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 545-576Crossref PubMed Scopus (1147) Google Scholar) and the observation that within F-actin there is an apparent opening of the cleft associated with ATP hydrolysis and phosphate release (14Belmont L.D. Orlova A. Drubin D.G. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 29-34Crossref PubMed Scopus (135) Google Scholar). The interactions of actin-binding proteins such as profilin and cofilin are regulated by the nucleotide bound to actin, and because these proteins are not expected to directly contact the nucleotide, they must sense the state of actin itself that is a function of the nucleotide bound. For example, the observation that ADF (actin-depolymerizing factor) binds G- or F-actin containing bound ADP with an affinity 2 orders of magnitude higher than it binds G- or F-actin containing bound ATP or ADP·Pi (15Carlier M.F. Laurent V. Santolini J. Melki R. Didry D. Xia G.X. Hong Y. Chua N.H. Pantaloni D. J. Cell Biol. 1997; 136: 1307-1322Crossref PubMed Scopus (817) Google Scholar) may be most simply explained by a conformational change within actin between these two nucleotide states. Consistent with this expectation, one study reported that the DNase I-binding loop in subdomain 2 of actin, which is a β-strand in one crystal (3Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1516) Google Scholar) and disordered in several other crystals (16McLaughlin P.J. Gooch J.T. Mannherz H.G. Weeds A.G. Nature. 1993; 364: 685-692Crossref PubMed Scopus (492) Google Scholar), becomes an α-helix when ADP rather than ATP is bound to actin (17Otterbein L.R. Graceffa P. Dominguez R. Science. 2001; 293: 708-711Crossref PubMed Scopus (407) Google Scholar). A more recent study has argued that there are no large conformational changes or domain shifts when ATP and ADP are bound to G-actin based upon the comparison of crystal structures for these two states (18Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Because solution data from the same work show a very large difference in the subtilisin digestion rates between ADP-G-actin and ATP-G-actin (18Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), it is possible that crystal packing interactions favor a closed state of G-actin even though in solution the state of the cleft may be shifted by the bound nucleotide. This type of conclusion appeared in another recent actin crystal structure study (19Klenchin V.A. Khaitlina S.Y. Rayment I. J. Mol. Biol. 2006; 362: 140-150Crossref PubMed Scopus (41) Google Scholar), which showed that cleavage of the DNase I-binding loop introduces no significant change in the crystal structure of G-actin, whereas complementary solution studies suggested that the cleavage is associated with an opening of the nucleotide-binding cleft. An analysis of multiple Arp2 (actin-related protein-2), Arp3, and actin crystal structures with different bound nucleotides also demonstrated that there are "inconsistencies" between the structures observed and simple nucleotide-dependent states (20Nolen B.J. Pollard T.D. Mol. Cell. 2007; 26: 449-457Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), suggesting that "alternative conformations have similar energies and are readily interconverted." Such a possibility is also advanced by a recent molecular dynamics study of nucleotide-dependent states of G-actin, which favors the helix-coil transition in the DNase I-binding loop (21Zheng X. Diraviyam K. Sept D. Biophys. J. 2007; 93: 1277-1283Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A comparison between actin and three actin-like molecules (Fig. 1) shows that although the nucleotide-binding cleft is relatively closed in all actin crystals (the opening that has been seen in one actin crystal (12Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (183) Google Scholar) is still small compared with the opening seen in actin-like proteins) in archaeal hexokinase (22Nishimasu H. Fushinobu S. Shoun H. Wakagi T. J. Biol. Chem. 2007; 282: 9923-9931Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), ADP can be bound to only the second major domain when the cleft is wide open (Fig. 1, upper left), so the phosphates are not making any contact with the first domain. More recent results suggest that ATP can be bound to the archaeal hexokinase when the major domains are in the same wide-open conformation. 2T. Wakagi, personal communication. A similar conclusion was reached about Arp2, for which it was shown that the second major domain can bind nucleotide in crystals even though the first major domain is disordered (20Nolen B.J. Pollard T.D. Mol. Cell. 2007; 26: 449-457Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The conclusion from this observation was that most of the binding energy for the nucleotide comes from the interaction with only the second major domain of Arp2. However, it would be expected for all proteins in this superfamily that ATP will be hydrolyzed only when the subunit is relatively closed, so the terminal phosphate will be in contact with the catalytic residues that are expected to be present in the first major domain. At this point, however, we have no detailed picture of the catalytic mechanism. In a human hexokinase I crystal structure (23Aleshin A.E. Kirby C. Liu X. Bourenkov G.P. Bartunik H.D. Fromm H.J. Honzatko R.B. J. Mol. Biol. 2000; 296: 1001-1015Crossref PubMed Scopus (82) Google Scholar), the two domains are more closed, and the β-phosphate within ADP is bound to the first domain (Fig. 1, upper right). Bacterial ParM was crystallized in two different conformations: in a closed state with ADP bound and in an open state with no nucleotide (8van den Ent F. Moller-Jensen J. Amos L.A. Gerdes K. Löwe J. EMBO J. 2002; 21: 6935-6943Crossref PubMed Scopus (203) Google Scholar). Interestingly, the relative domain orientations in the "open" state of ParM (with no nucleotide) (Fig. 1, lower left) are quite similar to those in the "closed" state of human hexokinase I (with ADP bound) (Fig. 1, upper right). What do these observations suggest? First, bacterial ParM cannot polymerize (24Garner E.C. Campbell C.S. Mullins R.D. Science. 2004; 306: 1021-1025Crossref PubMed Scopus (215) Google Scholar) under the conditions used for crystallization (no nucleotide or ADP), and these two conditions lead to very different crystal structures (8van den Ent F. Moller-Jensen J. Amos L.A. Gerdes K. Löwe J. EMBO J. 2002; 21: 6935-6943Crossref PubMed Scopus (203) Google Scholar). ParM requires ATP or an ATP analog for polymerization. In contrast, actin crystal structures are remarkably similar whether ADP or ATP is present, and actin can polymerize under all of these conditions (with ADP, ATP, or no nucleotide) (25Kasai M. Nakano E. Oosawa F. Biochim. Biophys. Acta. 1965; 94: 494-503Crossref PubMed Scopus (71) Google Scholar), albeit at different rates and with different critical concentrations for the polymerization. Concluding that G-actin appears to require only small conformational changes to polymerize, which we discuss below, it is possible that much larger conformational changes are associated with ParM binding of ATP, the ligand needed for polymerization to occur. An obvious question, which awaits an answer, is whether such small changes in G-actin, as reported from the crystal structures, can account for the nucleotide-dependent differences in actin affinity for actin-binding proteins. Although ParM and hexokinase can exist in the absence of bound nucleotide, actin is quite unstable unless a nucleotide is bound (26De La Cruz E.M. Pollard T.D. Biochemistry. 1995; 34: 5452-5461Crossref PubMed Scopus (59) Google Scholar). A model has been generated suggesting that the main role of the CCT chaperone needed for the proper folding of actin is to bring the two major domains together (27McCormack E.A. Llorca O. Carrascosa J.L. Valpuesta J.M. Willison K.R. J. Struct. Biol. 2001; 135: 198-204Crossref PubMed Scopus (27) Google Scholar), and in this model, the stability of the molecule is then maintained after folding by the bound nucleotide. Under conditions in which actin will crystallize (high protein and salt concentrations), it will also readily polymerize. The symmetry of an actin polymer (∼2.17 subunits/turn of a helix) is incompatible with any crystal space group (which can accommodate only helices with exactly two, three, four, or six subunits/turn), so G-actin has been either complexed with other proteins (such as profilin (28Schutt C.E. Myslik J.C. Rozycki M.D. Goonesekere N.C.W. Lindberg U. Nature. 1993; 365: 810-816Crossref PubMed Scopus (593) Google Scholar), DNase I (3Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1516) Google Scholar), and gelsolin (16McLaughlin P.J. Gooch J.T. Mannherz H.G. Weeds A.G. Nature. 1993; 364: 685-692Crossref PubMed Scopus (492) Google Scholar)) or drugs (such as latrunculin (29Bubb M.R. Govindasamy L. Yarmola E.G. Vorobiev S.M. Almo S.C. Somasundaram T. Chapman M.S. Agbandje-McKenna M. McKenna R. J. Biol. Chem. 2002; 277: 20999-21006Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 30Kudryashov D.S. Sawaya M.R. Adisetiyo H. Norcross T. Hegyi G. Reisler E. Yeates T.O. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13105-13110Crossref PubMed Scopus (46) Google Scholar), macrolides (31Allingham J.S. Zampella A. D'Auria M.V. Rayment I. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14527-14532Crossref PubMed Scopus (69) Google Scholar), and pectenotoxins (32Allingham J.S. Miles C.O. Rayment I. J. Mol. Biol. 2007; 371: 959-970Crossref PubMed Scopus (61) Google Scholar)) or changed in various ways (such as by mutation (18Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) or chemical modification (17Otterbein L.R. Graceffa P. Dominguez R. Science. 2001; 293: 708-711Crossref PubMed Scopus (407) Google Scholar)) to prevent polymerization during crystallization. As a result, we have no atomic resolution structure for F-actin, only models. The most important model that has been generated used a rotational and translational search for a placement of the G-actin crystal structure into a helical filament so as to best match the observed x-ray fiber diffraction pattern from an oriented F-actin gel (33Holmes K.C. Popp D. Gebhard W. Kabsch W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1306) Google Scholar). The "Holmes model" for F-actin generated by this procedure contains the explicit assumption that no large scale conformational change is needed between a G-actin monomer and an F-actin protomer. The only change in actin structure that was introduced involved the movement of a hydrophobic loop from the body of the subunit to form a contact with subunits on the opposite long-pitch helical strand. The absence of large conformational changes between the G-actin monomer and F-actin protomer has been confirmed by a number of low resolution electron microscopic (EM) 3The abbreviations used are:EMelectron microscopicFH2formin homology 2AMP-PNPadenosine 5′-(β,γ-imino)triphosphate. studies (34Orlova A. Egelman E.H. J. Mol. Biol. 1993; 232: 334-341Crossref PubMed Scopus (171) Google Scholar, 35Bremer A. Henn C. Goldie K.N. Engel A. Smith P.R. Aebi U. J. Mol. Biol. 1994; 242: 683-700Crossref PubMed Scopus (67) Google Scholar) as well as by cross-linking studies (36Kim E. Wriggers W. Phillips M. Kokabi K. Rubenstein P.A. Reisler E. J. Mol. Biol. 2000; 299: 421-429Crossref PubMed Scopus (56) Google Scholar, 37Orlova A. Galkin V.E. VanLoock M.S. Kim E. Shvetsov A. Reisler E. Egelman E.H. J. 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Cell Biol. 2001; PubMed Scopus Google Scholar). The for the binding of cofilin to F-actin suggested to from a within F-actin W. La Cruz E.M. J. Mol. Biol. 2006; PubMed Scopus Google Scholar, L. Pollard T.D. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), and the change of A. Egelman E.H. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) within an actin filament might be the for the binding of the (which actin to F-actin have been observed La Cruz E.M. Pollard T.D. Biochemistry. 1996; PubMed Scopus Google Scholar, La Cruz E.M. Pollard T.D. Biochemistry. 1994; PubMed Scopus Google Scholar), suggesting that such might the binding of a number of drugs and proteins to The in F-actin and the dynamics of actin are in the ADP state compared with the ATP and ADP·Pi states. these differences to a of subdomain 2 interactions with the protomer in as by the A. P. C. Reisler E. J. Biol. 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Cell. 2007; 26: 449-457Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and hexokinase (22Nishimasu H. Fushinobu S. Shoun H. Wakagi T. J. Biol. Chem. 2007; 282: 9923-9931Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, A.E. C. Bartunik H.D. Fromm H.J. Honzatko R.B. J. Mol. Biol. 282: PubMed Scopus Google Scholar) that nucleotide can be bound only to the second major domain of proteins in this What are the of these different within The many other proteins that bind F-actin require and these different but thus only cofilin has been seen to directly one of these in F-actin V.E. Orlova A. VanLoock M.S. Shvetsov A. Reisler E. Egelman E.H. J. Cell Biol. PubMed Scopus Google Scholar). such and in F-actin are important is also by the that actin and in actin by E. Biochemistry. 1997; PubMed Scopus Google Scholar, E. E. Orlova A. Hegyi G. Egelman E.H. A. Reisler E. Biochemistry. PubMed Scopus Google Scholar). 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Biol. 2002; 12: 570-575Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), are highly in in contacts in F-actin the of the we a that in solution at atomic resolution with a time all of the questions that still exist about the structure and dynamics of actin would be readily Indeed, the study of all would be quite different from what it is the in molecular the no such is on the with the more available to we think it that some of the most important questions about actin can still be in the questions will be by higher resolution structures of the actin by and and by for the of actin N. H. K. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar).