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• W1966349701 abstract "Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics. Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics. The cryo-EM structure of actin filament reveals intermolecular interactions Mg2+ and phosphate ions (Pi) play an important role in stabilizing the filament The bending of the Pro-rich loop triggers the activation of the ATPase activity A cavity in the catalytic site plays a role in releasing hydrolyzed γ-phosphates The actin-filament system is required in almost all cytoplasmic processes, including cell adhesion, motility, cellular signaling, intracellular trafficking, and cytokinesis. Although stable actin filaments (F-actin) are necessary during muscle contraction, the active turnover of filaments is required in many cell functions. Actin has two major domains separated by a nucleotide-binding cleft (Kabsch et al., 1990Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Atomic structure of the actin:DNase I complex.Nature. 1990; 347: 37-44Crossref PubMed Scopus (1491) Google Scholar). The outer domain is divided into subdomains 1 and 2 and the inner domain into subdomains 3 and 4. All of the subdomains interact with the bound nucleotide. ATP is hydrolyzed at the rate of 1/3.3 s−1 following the elongation of filaments at the growing end of filaments (Blanchoin and Pollard, 2002Blanchoin L. Pollard T.D. Hydrolysis of ATP by polymerized actin depends on the bound divalent cation but not profilin.Biochemistry. 2002; 41: 597-602Crossref PubMed Scopus (130) Google Scholar), whereas the phosphate release is 100 times slower (Carlier and Pantaloni, 1986Carlier M.F. Pantaloni D. Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments.Biochemistry. 1986; 25: 7789-7792Crossref PubMed Scopus (102) Google Scholar). As a result, newly polymerized filaments consist of stable ADP-Pi actin (abbreviated as F-ADP-Pi), whereas the older filaments contain mainly ADP actin (F-ADP), which disassembles more rapidly (Carlier and Pantaloni, 1986Carlier M.F. Pantaloni D. Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments.Biochemistry. 1986; 25: 7789-7792Crossref PubMed Scopus (102) Google Scholar). Under physiological conditions, inorganic phosphate (Pi) binds to F-actin and reduces the critical concentration for polymerization (Rickard and Sheterline, 1986Rickard J.E. Sheterline P. Cytoplasmic concentrations of inorganic phosphate affect the critical concentration for assembly of actin in the presence of cytochalasin D or ADP.J. Mol. Biol. 1986; 191: 273-280Crossref PubMed Scopus (41) Google Scholar, Fujiwara et al., 2007Fujiwara I. Vavylonis D. Pollard T.D. Polymerization kinetics of ADP- and ADP-Pi-actin determined by fluorescence microscopy.Proc. Natl. Acad. Sci. USA. 2007; 104: 8827-8832Crossref PubMed Scopus (131) Google Scholar). Actin dynamics also depends on the identity of the bound divalent cation, physiologically Mg2+, associated with the bound nucleotide (Carlier et al., 1986Carlier M.F. Pantaloni D. Korn E.D. Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin.J. Biol. Chem. 1986; 261: 10778-10784Abstract Full Text PDF PubMed Google Scholar). Although a vast amount of biochemical data has been accumulated, the quest for a definitive and detailed molecular mechanism of the polymerization of monomeric actin (G-actin) to filamentous actin (F-actin) has been hampered by the inherent flexibility of actin filament. The flexibility has not allowed an atomic structure of F-actin to be determined. More than 50 atomic structures of G-actin bound with ATP or ADP have been determined since 1990 (Kabsch et al., 1990Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Atomic structure of the actin:DNase I complex.Nature. 1990; 347: 37-44Crossref PubMed Scopus (1491) Google Scholar), but F-actin has been visualized to relatively moderate resolution either by three-dimensional (3D) image reconstruction from electron micrographs (Belmont et al., 1999Belmont L.D. Orlova A. Drubin D.G. Egelman E.H. A change in actin conformation associated with filament instability after Pi release.Proc. Natl. Acad. Sci. USA. 1999; 96: 29-34Crossref PubMed Scopus (135) Google Scholar) or modeling based on X-ray fiber diagrams (Holmes et al., 1990Holmes K.C. Popp D. Gebhard W. Kabsch W. Atomic model of the actin filament.Nature. 1990; 347: 44-49Crossref PubMed Scopus (1286) Google Scholar, Lorenz et al., 1993Lorenz M. Popp D. Holmes K.C. Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm.J. Mol. Biol. 1993; 234: 826-836Crossref PubMed Scopus (441) Google Scholar). The inherent flexibility of actin filaments hampers determination of atomic structure. Recently, a new model of F-actin based on improved X-ray fiber diffraction analysis was reported (Oda et al., 2009Oda T. Iwasa M. Aihara T. Maéda Y. Narita A. The nature of the globular- to fibrous-actin transition.Nature. 2009; 457: 441-445Crossref PubMed Scopus (426) Google Scholar). Oda et al. proposed that outer-domain movement upon assembly flattens the actin molecule in the polymer, similar to the case of the bacterial actin homolog MreB (van den Ent et al., 2001van den Ent F. Amos L.A. Löwe J. Prokaryotic origin of the actin cytoskeleton.Nature. 2001; 413: 39-44Crossref PubMed Scopus (593) Google Scholar), and that the DNase I binding loop (DNase I loop) adopts an open loop conformation. However, the mechanism of ATP hydrolysis and its coupling with actin assembly remains poorly understood. Here, we present cryo-electron microscopic (cryo-EM) data in which single-particle analysis has been applied to short and relatively straight stretches of filaments, with Pi added in the millimolar range (similar to the intracellular Pi concentration), to further minimize filament flexibility (Nonomura et al., 1975Nonomura Y. Katayama E. Ebashi S. Effect of phosphates on the structure of the actin filament.J. Biochem. 1975; 78: 1101-1104Crossref PubMed Scopus (33) Google Scholar). The quality of the cryo-EM images was further refined as described in the Experimental Procedures. The resolution of the final reconstruction was estimated to be ∼5 Å (Fourier shell correlation [FSC] of 0.143 at 4.7 Å, a criterion according to Rosenthal and Henderson, 2003Rosenthal P.B. Henderson R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy.J. Mol. Biol. 2003; 333: 721-745Crossref PubMed Scopus (1121) Google Scholar) or ∼8 Å (FSC of 0.5 at 7.8 Å, a traditional criterion), and some of the α-helical backbones and large side chains can be directly observed. This indicates that the data quality was sufficient to visualize the structural changes upon polymerization and allowed us to build a quasi-atomic model of F-actin (F-ADP+Pi). Putative Mg2+-binding sites and Pi-binding sites of F-actin, which play an important role in actin assembly, were identified in the EM map, and the proline (Pro)-rich loop (residues 108–112) was observed to adopt a more bent configuration that would trigger a phosphate-releasing pathway. Crystal structures of G-actin with mutations in this loop, in which the ATPase activity was increased or decreased, further revealed the region required for Pi release (the so-called back-door region; Wriggers and Schulten, 1999Wriggers W. Schulten K. Investigating a back door mechanism of actin phosphate release by steered molecular dynamics.Proteins. 1999; 35: 262-273Crossref PubMed Scopus (95) Google Scholar) and the atomic details of the mechanism of ATP hydrolysis. The combined structural information sheds new light on the coupling mechanism of ATP hydrolysis and F-actin assembly. The 3D cryo-EM structure was reconstructed from segments containing 26 actin molecules (Figure 1B ). Approximately 8000 actin molecules from zero energy-loss cryo-EM images of actin filaments in the presence of phosphate (Figure 1A) contributed to the final EM map (Figure 1 and Figure S1 available online). A quasi-atomic model (Figures 1B and 1D) was constructed by refining the initial F-actin model consisting of 26 G-ADP actin molecules (Rould et al., 2006Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) to obtain a good fit into the EM density map (see Figure S1, Movie S1, Movie S2, and Figure 1E for FSC and figure-of-merit [FOM] plots). The resolution of the structure of F-actin appeared to be nonuniform depending on the regions (Figure 1E). In the region where three actin molecules interact within the filament, the quality of EM map was better (Figure 1E) and the backbone structure of α helices 5 and 6 (h5, residues 183–196; h6, residues 207–216) and the Thr-rich loop (residues 197–204) could be clearly resolved, allowing the assignment of some large side chains such as Lys191, Tyr198, and Arg206 (Figures 1C and 1D). Although no β structure could be directly visualized, most α helices and loops defined in Figure 2C could be assigned. The N-terminal segment (residues 1–5), h0 (residues 41–48) in the middle of DNase I loop, and h7 (mobile helix: residues 226–230) were less clearly resolved but still allowed main-chain placement except for the h0 segment, which is disordered. The structural details are shown in Figures S1F–S1H.Figure S1Assessment of Resolution and the Model Building, Related to Figure 1Show full caption(A and C) The refined atomic structure of F-ADP+Pi (magenta, blue, green for each actin molecule). The region for the intermolecular interaction is shown with EM density (gray contours) and a phosphate ion at phosphate-binding site 1 (Pi1, orange sphere) and a magnesium ion at magnesium-binding site 1 (gray sphere) are also shown. The α-helical torsion and some side chains are visible in the EM map, and the atomic model is well fitted into the EM densities. The backbone of G-ADP structure (gray stick) is also superimposed onto the EM density map.(B and D) The corresponding electron densities of the crystal structure of actin mutant P109I were generated at the resolution of 4, 5, 6, or 8 Å. The α-helical torsion and some side chains are visible at 4 or 5 Å resolution maps, whereas the 6 or 8 Å resolution electron density map lacks those features.(E) Conformational changes of actin in the intermolecular interface. Rotated by ∼90° with respect to (A). The F-ADP+Pi structure (magenta, blue, green) and the G-ADP structure (Rould et al., 2006Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) (gray) are shown in the left and right panels, respectively, with the EM density (gray contours).(F–H) The EM densities for the DNase I loop (F), the helix 7 and the Ser-rich loop (residues 231–237) in subdomain 4 (G), and the N-terminal region (H). The main chains could be traced except for the middle part (residues 41–48) of the DNase I loop. The B-factor correction was not applied to these EM maps.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Comparison of F-Actin Structure with G-Actin StructureShow full caption(A) The inner domain of the G-actin (G-ADP) (gray; Otterbein et al., 2001Otterbein L.R. Graceffa P. Dominguez R. The crystal structure of uncomplexed actin in the ADP state.Science. 2001; 293: 708-711Crossref PubMed Scopus (397) Google Scholar) was superimposed onto that of actin filament (F-actin) (green). In the F-actin structure (F-ADP+Pi), the outer domain is rotated by ∼16° relative to the inner domain. The rotation angle was determined using DynDom (Hayward and Berendsen, 1998Hayward S. Berendsen H.J.C. Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme.Proteins. 1998; 30: 144-154Crossref PubMed Scopus (685) Google Scholar). The bound ADP is shown in ball and stick format.(B) The enlarged frontal view of the hydrophobic cleft in F-actin (green) and G-actin (gray). DNase I loop (not shown) fits in the rear half of hydrophobic cleft. The front half of hydrophobic cleft remains empty and widens. The double-headed arrows show that the distance betweenTyr143 and Leu346 is wider in F-actin compared with that in G-actin. The arrow indicates the hinge point of the outer-domain rotation.(C) Root-mean-square deviation (rmsd) per residue between the molecules of F-actin and G-actin. For the calculation, each of the inner and outer domains was superimposed onto that of G-actin independently. The structure within each domain is essentially the same as that of G-actin. The conformational changes occur mainly in DNase I loop, Pro-rich loop, Thr-rich loop, mobile helix h7, and Ser-rich loop, all of which are involved in the intermolecular interfaces.(D) Actin polymerization assay. Actin mutants (2.3 μM) were incubated at 25°C, ultracentrifuged, and analyzed by SDS-PAGE. The data represent mean values with standard errors of the actin pellets (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and C) The refined atomic structure of F-ADP+Pi (magenta, blue, green for each actin molecule). The region for the intermolecular interaction is shown with EM density (gray contours) and a phosphate ion at phosphate-binding site 1 (Pi1, orange sphere) and a magnesium ion at magnesium-binding site 1 (gray sphere) are also shown. The α-helical torsion and some side chains are visible in the EM map, and the atomic model is well fitted into the EM densities. The backbone of G-ADP structure (gray stick) is also superimposed onto the EM density map. (B and D) The corresponding electron densities of the crystal structure of actin mutant P109I were generated at the resolution of 4, 5, 6, or 8 Å. The α-helical torsion and some side chains are visible at 4 or 5 Å resolution maps, whereas the 6 or 8 Å resolution electron density map lacks those features. (E) Conformational changes of actin in the intermolecular interface. Rotated by ∼90° with respect to (A). The F-ADP+Pi structure (magenta, blue, green) and the G-ADP structure (Rould et al., 2006Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) (gray) are shown in the left and right panels, respectively, with the EM density (gray contours). (F–H) The EM densities for the DNase I loop (F), the helix 7 and the Ser-rich loop (residues 231–237) in subdomain 4 (G), and the N-terminal region (H). The main chains could be traced except for the middle part (residues 41–48) of the DNase I loop. The B-factor correction was not applied to these EM maps. (A) The inner domain of the G-actin (G-ADP) (gray; Otterbein et al., 2001Otterbein L.R. Graceffa P. Dominguez R. The crystal structure of uncomplexed actin in the ADP state.Science. 2001; 293: 708-711Crossref PubMed Scopus (397) Google Scholar) was superimposed onto that of actin filament (F-actin) (green). In the F-actin structure (F-ADP+Pi), the outer domain is rotated by ∼16° relative to the inner domain. The rotation angle was determined using DynDom (Hayward and Berendsen, 1998Hayward S. Berendsen H.J.C. Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme.Proteins. 1998; 30: 144-154Crossref PubMed Scopus (685) Google Scholar). The bound ADP is shown in ball and stick format. (B) The enlarged frontal view of the hydrophobic cleft in F-actin (green) and G-actin (gray). DNase I loop (not shown) fits in the rear half of hydrophobic cleft. The front half of hydrophobic cleft remains empty and widens. The double-headed arrows show that the distance betweenTyr143 and Leu346 is wider in F-actin compared with that in G-actin. The arrow indicates the hinge point of the outer-domain rotation. (C) Root-mean-square deviation (rmsd) per residue between the molecules of F-actin and G-actin. For the calculation, each of the inner and outer domains was superimposed onto that of G-actin independently. The structure within each domain is essentially the same as that of G-actin. The conformational changes occur mainly in DNase I loop, Pro-rich loop, Thr-rich loop, mobile helix h7, and Ser-rich loop, all of which are involved in the intermolecular interfaces. (D) Actin polymerization assay. Actin mutants (2.3 μM) were incubated at 25°C, ultracentrifuged, and analyzed by SDS-PAGE. The data represent mean values with standard errors of the actin pellets (n = 4). In the F-actin structure, the relation between the two major actin domains is different from that in G-actin. The outer domain is found rotated in a swing-door manner by ∼16° relative to the inner domain (Figure 2A). The pivoting point, Asp154 next to P loop 2, is located near the bound nucleotide, with two hinges: Gln137-Ala138 and Lys336-Tyr337 (Figures 2A and 2C). The axis of the rotation was oriented by ∼40° relative to the filament helix axis (Figure 2A) and not vertical to the helix axis (Oda et al., 2009Oda T. Iwasa M. Aihara T. Maéda Y. Narita A. The nature of the globular- to fibrous-actin transition.Nature. 2009; 457: 441-445Crossref PubMed Scopus (426) Google Scholar) (Figure S2). The outer-domain rotation enables the DNase I loop to fit in the rear half of the hydrophobic cleft (Figure 1F) so that it could reach Leu110, which can be clearly assigned in the EM map, of the actin molecule on the minus-end side (Figure 1C and Movie S1). As a result of the outer-domain rotation (Figure 2A), the front half of the hydrophobic cleft is widened, making the side chain of Tyr143 more solvent exposed and increasing the distance between Tyr143 and Leu346 on the hydrophobic helix (h12) (Figure 2B). The importance of the front half of the hydrophobic cleft for polymerization is further highlighted by the fact that the Dictyostelium actin with Tyr143Phe mutation polymerizes poorly. The Tyr143Ile mutation, however, has only a small inhibitory effect on assembly (Figure 2D). This is consistent with the fact that the corresponding residue of the bacterial actin homolog MreB is Ile (van den Ent et al., 2001van den Ent F. Amos L.A. Löwe J. Prokaryotic origin of the actin cytoskeleton.Nature. 2001; 413: 39-44Crossref PubMed Scopus (593) Google Scholar). The front half of the hydrophobic cleft is also a primary site for G-actin- and/or F-actin-binding proteins, which could regulate actin assembly by promoting or blocking the widening of the hydrophobic cleft. Indeed, small marine toxins such as kabirimide C and jaspisamide A (Klenchin et al., 2003Klenchin V.A. Allingham J.S. King R. Tanaka J. Marriott G. Rayment I. Trisoxazole macrolide toxins mimic the binding of actin-capping proteins to actin.Nat. Struct. Biol. 2003; 10: 1058-1063Crossref PubMed Scopus (120) Google Scholar), which bind to the front half of the hydrophobic cleft and sever actin filament, are sterically compatible with our F-actin structure. This may partially account for the inhibitory effect of modification of Cys374 with tetramethylrhodamine (TMR) on polymerizability (Otterbein et al., 2001Otterbein L.R. Graceffa P. Dominguez R. The crystal structure of uncomplexed actin in the ADP state.Science. 2001; 293: 708-711Crossref PubMed Scopus (397) Google Scholar). Accompanying the domain rotation, the main-chain atoms shift more than 4.8 Å mainly in the loop regions (Figure 2C), which facilitates intermolecular interactions. This results in a buried surface area between three actin molecules of up to 7646 Å2, which is a substantial increase compared to 2998 Å2 for the crystal structure of G-ADP actin docked into the EM map without remodeling. At the interface of the three actins within the filament (Figures 1C and 1D and Figure 3A ), the Thr-rich loop (containing Thr202 and Thr203) was remarkably different from that in G-actin (shown in gray in Figure 3A). Furthermore, the N-terminal part of h6 was shifted compared to that in G-actin (shown in gray in Figure 3A and Figure S1A) and possibly stabilized by a putative salt bridge of Glu205 with Arg290 of the upper molecule and the hydrophobic interaction of Ala204 and Ile208 with Ile287 of the upper molecule. The disruption of h6 is observed in the crystal structure of the actin-DNase I complex (Kabsch et al., 1990Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Atomic structure of the actin:DNase I complex.Nature. 1990; 347: 37-44Crossref PubMed Scopus (1491) Google Scholar). Local reordering of the N-terminal part of h6 could provide a common binding site for actin-binding proteins, including actin itself, and could also help bind a magnesium ion (site 1 in Figure 3A) and two phosphate ions (Pi1 and Pi2, Figure 1D). This feature of h6 is supported by Dictyostelium mutants Val287Asp or Arg290Glu, which both polymerized poorly (Figure 3G), emphasizing the importance of Ile287 (Val287 in Dictyostelium actin) and Arg290 in the vertical interaction. In addition, these mutants exhibit more disperse distribution when fused to GFP in Dictyostelium cells, with the double mutant displaying a more prominent phenotype (Figure 3F). Both of two Pi-binding loops, P loop 1 (residues 13–16) and P loop 2 (residues 156–159), could be assigned (Figure 4B ). They surrounded the densities that correspond to α- and β-phosphates of the bound nucleotide, with no evidence for any γ-phosphate density. The region, where the γ-phosphate is located in the ATP form, is occupied by bulk solvent (Figure 4B), indicating that F-actin has bound ADP in the ATPase site (Figure 4B). Similar to the majority of crystal structures of G-actin with ATP or ADP, the nucleotide-binding cleft (Nolen and Pollard, 2007Nolen B.J. Pollard T.D. Insights into the influence of nucleotides on actin family proteins from seven structures of Arp2/3 complex.Mol. Cell. 2007; 26: 449-457Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) is closed. However, the P loop 1 encircles a low-density region (a bubble in the EM map; Figure S4A ), suggesting that two strands, s1 (residues 8–11) and s2 (residues 16–18), might be dynamically deformed. This could allow accommodation of the outer-domain rotation. Consistent with this, NMR spectroscopy showed that residues 1–22, which include P loop 1, are mobile even in an F-actin state (Heintz et al., 1996Heintz D. Kany H. Kalbitzer H.R. Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by 1H and 19F nuclear magnetic resonance spectroscopy.Biochemistry. 1996; 35: 12686-12693Crossref PubMed Scopus (24) Google Scholar).Figure S4Cross-Sections of the F-Actin Map to Show the Relationship between ADP, Cavity, and Phosphate-Binding Site 1, Related to Figure 4Show full caption(A and B) Top view of the cross section (A) and side view of vertical section (B) of F-actin in stereo. A B-factor correction was not applied to these EM maps (blue contour). The cylindrical intermolecular cavity (∼18 Å long, ∼6 Å in diameter) is formed along the groove between the outer and inner domains of the green molecule and flanked by the subdomain 4 of the purple actin molecule. The hydrolyzed γ-phosphate will gain access to the external solvent only through this cavity. Because the phosphate-binding site 1 (Pi1) is located near the exit of the cavity, the hydrolyzed phosphate will be bound to this site before it escapes to the external solvent. A low-density region labeled as “bubble” is located near Gly13 and Leu16.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) Top view of the cross section (A) and side view of vertical section (B) of F-actin in stereo. A B-factor correction was not applied to these EM maps (blue contour). The cylindrical intermolecular cavity (∼18 Å long, ∼6 Å in diameter) is formed along the groove between the outer and inner domains of the green molecule and flanked by the subdomain 4 of the purple actin molecule. The hydrolyzed γ-phosphate will gain access to the external solvent only through this cavity. Because the phosphate-binding site 1 (Pi1) is located near the exit of the cavity, the hydrolyzed phosphate will be bound to this site before it escapes to the external solvent. A low-density region labeled as “bubble” is located near Gly13 and Leu16. The sensor loop (residues 71–77), which reflects the state of bound nucleotide of G-actin (Rould et al., 2006Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), adopts a conformation similar to that of G-ADP (Otterbein et al., 2001Otterbein L.R. Graceffa P. Dominguez R. The crystal structure of uncomplexed actin in the ADP state.Science. 2001; 293: 708-711Crossref PubMed Scopus (397) Google Scholar, Rould et al., 2006Rould M.A. Wan Q. Joel P.B. Lowey S. Trybus K.M. Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states.J. Biol. Chem. 2006; 281: 31909-31919Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) (Figure 3A). Compared with G-ATP actin, Glu72 on the sensor loop moves upwards by 2 Å and closer to Arg183. This enables a putative salt bridge between Glu72 and Arg183, which stabilize F-actin, as Arg183 is also associated with two phosphate ions. Actin is known to have several binding sites for cations and phosphates (Rickard and Sheterline, 1986Rickard J.E. Sheterline P. Cytoplasmic concentrations of inorganic phosphate affect the critical concentration for assembly of actin in the presence of cytochalasin D or ADP.J. Mol. Biol. 1986; 191: 273-280Crossref PubMed Scopus (41) Google Scholar, Carlier et al., 1986Carlier M.F. Pantaloni D. Korn E.D. Fluorescence measurements of the binding of cations to high-affinity and low-affinity sites on ATP-G-actin.J. Biol. Chem. 1986; 261: 10778-10784Abstract Full Text PDF PubMed Google Scholar). Also, more than 50 crystal structures of G-actin have been determined, and magnesium-binding sites have been deduced (Table 1; Klenchin et al., 2006Klenchin V.A. Khaitlina S.Y. Rayment I. Crystal structure of polymerization-competent actin.J. Mol. Biol. 2006; 362: 140-150Crossref PubMed Scopus (41) Google Scholar).Table 1Putative Phosphates and Magnesium Ions in F-Actin and Corresponding Sites in Crystal Structures of G-ActinSites in F-ActinPeak HeightaPeak height in the EM difference map (EM density minus the atomic model).Coordinating Residues in F-ActinCorresponding Sites in G-Actin with the PDB Code1Pi (site 1)5.2 σR183, D184, T202bThe residues in the longitudinally and obliquely located actin molecules are indicated in underline and italic, respectively., K2843CI5 (SO42−), 2Q36 (SO42−), 1YAG (SO42−), 1D4X (SO42−), 1YVN (SO42−), 1NLV (SO42−), 1NM1 (SO42−), 1NMD (SO42−)2Pi (site 2)5.9 σR183, R2063CI5 (SO42−)3Pi" @default.
• W1966349701 created "2016-06-24" @default.
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• W1966349701 date "2010-10-01" @default.
• W1966349701 modified "2023-10-10" @default.
• W1966349701 title "Structural Basis for Actin Assembly, Activation of ATP Hydrolysis, and Delayed Phosphate Release" @default.
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