The structure of nonvertebrate actin: Implications for the ATP hydrolytic mechanism

Abstract

The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 Å. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.A ctin is a ubiquitous 43-kDa adenine nucleotide-binding protein involved in a wide range of fundamental processes in eukaryotic cells, including cell motility (1), cytokinesis (2), vesicle transport (3, 4), and the establishment and maintenance of cell morphology (5, 6). Actin is also an essential component of numerous specialized structures in higher eukaryotes, such as the intestinal brush border (7,8) and stereocilia (9, 10). The ability of actin to participate in these diverse processes is a consequence of the dynamic and polymorphic nature of the filamentous actin (F-actin) assemblies that are formed in vivo.Central to understanding cytoskeletal regulation is the contribution of the adenine nucleotide to actin structure, function, and dynamics. Bound nucleotide is required to stabilize the actin monomer under physiological conditions, because nucleotidefree actin denatures at a rate of 0.2 s Ϫ1 (11). ATP hydrolysis occurs subsequent to monomer addition to the growing filament, thus ''marking'' the oldest part of the filament (i.e., ADP-actin), and may serve as a timing mechanism for filament disassembly, analogous to the role of GTP hydrolysis in G proteins (12, 13). Furthermore, several regulatory proteins (e.g., profilin, cofilin, and thymosin ␤-4) show specificity for either ATP-or ADPactin, which is thought to be an important element of cytoskeletal regulation (12). Actin dynamics also depends on the identity of the bound metal-nucleotide complex (14-21).The structure of the actin monomer has been known for over a decade, and the structures of mammalian Ca 2ϩ -ATP actins in complex with DNase I (22), profilin (23), gelsolin segment-1 (24), and gelsolin segments-4-6 (25) are available. An orthorhombic crystal form of the Ca 2ϩ -ATP Dictyostelium actin͞ gelsolin segment-1 structure has also been reported (26). These structures highlighted specific regulatory interactions and revealed the remarkable architectural similarity between actin and a wide range of proteins (27), including the heat shock cognate ATPases (e.g., HSC70) and sugar kinases (e.g., hexokinase) (28-3...

Nucleotide-Mediated Conformational Changes of Monomeric Actin and Arp3 Studied by Molecular Dynamics Simulations

“…In the actin crystal structures, the calcium has a complete shell of hydration and does not directly contact any of the residues in the nucleotide cleft. 11,13 In these structures, the calcium is coordinated to the water in a heptagonal arrangement, with an average distance of 2.5 Å. We sought to approximate realistic behavior by lowering the charge on the calcium.…”

“…16 This valine is found in either an "up" or a "down" conformation in Arp3 and actin crystal structures. 13,21 With the side chain in the down position, the backbone amide of this residue can hydrogen bond to the γ-phosphate, but in the up position, it cannot. The up position of the valine may open up a path for phosphate to dissociate from the ADP-P i intermediate.…”

“…16 This residue has been observed in both up and down positions in actin and Arp3. 13,21 In the down position, its backbone amide hydrogen bonds to the γ-phosphate and its side chain blocks the "back door" for phosphate release as proposed. 28 We previously hypothesized that as the γ-phosphate moves away from the β-phosphate after hydrolysis, it stays hydrogen bonded to Val159/174 as this residue flips up to open the back door.…”

“…In most crystal structures of actin, the nucleotide binding cleft is closed around either ATP or ADP. [13][14][15][16][17][18] So far, the cleft is open only in certain crystals of ATP-β-actin bound to profilin, where the P1 and P2 loops were separated by an additional 3 Å from closed conformations. 14 However, the cleft was closed in crystals of the same complex grown in different conditions.…”

The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin

“…The 3D structure of monomeric actin (G-actin) was determined first by Kabsch et al (2) as a complex, with DNase I serving as a polymerization inhibitor. The crystal structure of monomeric actin has since been determined at atomic resolution under a variety of conditions, varying, for example, in actin isoform (3), the identity of the bound nucleotide (4,5), and in the identity of the other proteins (6,7) or small molecules added as polymerization inhibitors (8,9). As a consequence, the structure of the actin monomer (G-actin) is understood in considerable detail, although some important issues remain open regarding nucleotide-dependent changes in G-actin structure, including a possible transition between the open and closed nucleotide cleft conformations and the order-disorder shift of the DNase I-binding loop (4,7,10).…”