Structures and mechanisms of actin ATP hydrolysis

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) P^G–O^3B bond cleavage; 3) four concomitant events: W1–PO₃⁻ formation, OH⁻ and proton cleavage, nucleophilic attack by the OH⁻ against P^G, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi–bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.

Actin, a major cytoskeletal protein, is an ATP-binding protein that exists in monomeric (G-actin) and filamentous (F-actin) forms. A large variety of cellular functions are driven by the cyclic processes of actin molecule assembly (polymerization) and disassembly (depolymerization). Actin ATP hydrolysis, which was originally discovered without knowing its biological significance (1), energetically drives the cyclic assembly–disassembly (2). Therefore, the elucidation of the mechanism of ATP hydrolysis is crucially important for our understanding of the cellular functions of actin.The actin molecule comprises two major domains, the outer domain (OD) and the inner domain (ID). An ATP molecule with a divalent cation binds in the cleft between the OD and ID (3). Upon incorporation into the actin filament, the actin molecule undergoes a conformational transition (4). In the monomeric G-form, the OD is twisted by about 20° relative to the ID, whereas in the filamentous F-form the two domains are almost flat.The entire cycle of actin assembly–disassembly proceeds in five sequential processes (below, each nucleotide tightly binds Mg²⁺, which is not explicitly indicated for simplicity. -G and -F indicate G-form and F-form actin, respectively):

• 1)ATP-G → ATP-F: The conformational transition, which is associated with the polymerization of the ATP-bound G-form actin.
• 2)ATP-F → ADP-Pi-F: The ATP hydrolysis reaction, triggered by process 1 with a rate constant of 0.3 s⁻¹ (5), which is considerably slower than other ATPases [e.g., myosin (20 to 200 s⁻¹) (6)]. The slow rate which allows further elongation, and the hydrolysis reaction occurs in the interior of the filament. The reaction is irreversible (7).
• 3)ADP-Pi-F → ADP-F: The Pi release, which occurs at an extremely slow rate with actin molecules in the interior of the filament [0.003 s⁻¹ (8) or 0.007 s⁻¹ (9)] but is much faster with molecules around barbed ends [>2 s⁻¹ (10) or 1.8 s⁻¹ (9)]. The Pi release is reversible. ADP-F has a modest affinity for Pi with a dissociation constant of 1 mM (9, 10), which is far below the cytosolic concentration, whereas ADP-G hardly binds Pi (∼60 mM) (10).
• 4)ADP-F → ADP-G: The depolymerization releases actin subunits at the ends of the actin filament.
• 5)ADP-G → ATP-G: The bound nucleotide exchanges from ADP for ATP, which occurs only in G-actin.

The cyclic reaction of barbed end assembly and the pointed end disassembly, occurring in actin alone at the critical concentration of monomers using ATP as an energy source, is called tread-milling (11). In the cytosol, where the actin concentration is far beyond the critical concentration for polymerization, cooperative behaviors of regulatory proteins accelerate the spontaneous tread-milling and/or facilitate filament turnover by promoting multiple reactions involving nucleation, capping, and severing, to drive fast cell migration (12, 13). Recent progress, particularly in kinetic analyses of single actin filaments, has revealed the contribution of the barbed end disassembly to monomer recycling (14, 15). Pi release dramatically alters the assembly properties of actin filaments in two ways: 1) by promoting spontaneous depolymerization and, more importantly, 2) by making the ADP-actin subunits more attractive to proteins that facilitate filament disassembly such as ADF/cofilin. Therefore, for comprehensively understanding of the actin cycle of assembly–disassembly, the mechanism and significance of the Pi release are of crucially important. This should be based upon our knowledge of the actin ATP hydrolysis process and the properties of ADP-Pi-F.Studies of the actin ATP hydrolysis mechanism have been hampered, mainly due to the lack of F-form actin structures at a sufficient resolution for identifying water molecules, which are the keys to the reaction. The crystal structure of the G-form actin is not suitable (3, 16), since the G-form actin is not ATP hydrolysis–competent (17). The cryogenic electron microscopy (cryo-EM) structures of the actin filament at over 3.1-Å resolutions (18–20) are also insufficient, because the details of the water molecules in the nucleoside binding cleft, particularly those surrounding the phosphate moiety, are missing. The actin filament has not been crystallized, because filaments with a fixed length have never been prepared. Previously, the ATP hydrolysis mechanism of F-form actin was studied by employing metadynamics simulations (21). However, the details of the reaction mechanism have remained obscure, since the simulations were based upon structural data that lacked water molecules (4).We now show that, in a 1:1 complex of the actin-binding protein fragmin domain-1 (F1) and actin (referred to as the F1A complex), the actin molecule is trapped in the F-form. This is the first monomeric actin structure in the F-form among the more than 260 actin structures deposited to date in the Protein Data Bank (PDB) (22). Fragmin is a member of the villin-gelsolin protein superfamily (23) and was first isolated from the slime mold Physarum polycephalum (24). Like gelsolin, fragmin severs F-actin and caps the barbed end in a Ca²⁺-dependent manner (24–26). Fragmin consists of three tandemly linked domains (F1, F2, F3) that are homologous to the N-terminal half of gelsolin (G1, G2, G3). G1 also forms a 1:1 complex with actin. However, in the G1–actin complex, the actin is in the G-form (27).The F1A complex provided 1.15-Å resolution structures of F-form actin binding AMPPNP, ADP-Pi, or ADP, with a magnesium ion at the catalytic site. Based on the high precision of the pre- and posthydrolysis structures, we performed quantum mechanics/molecular mechanics (QM/MM) calculations (28), which revealed the comprehensive reaction mechanism of the actin ATP hydrolysis. The results provide mechanistic answers to key questions regarding the cycle of actin state transitions: how the G-to-F conformational transition triggers ATP hydrolysis, why the ATP hydrolysis rate is much slower than those of other ATPases, why the hydrolysis is irreversible, why the Pi release is so slow, and why F-form actin is destabilized after the Pi-release.Our results demonstrate that, while some properties are quantitatively different, the overall catalytic strategy of actin ATP hydrolysis (up until the formation of ADP-Pi-F) is almost identical to those of P-loop type-motor proteins, such as F₁-ATPase (29), kinesin (30), and myosin (31–33), despite the substantial differences in the catalytic site structures. In contrast to the common ATP hydrolysis strategies, the processes after the hydrolysis are distinct in actin: The hydrolysis does not cause any overall conformational changes; therefore, the Pi is not promptly released and the ADP-Pi F-form actin structure is readily available. These observations have led us to propose hypotheses about how ATP hydrolysis and Pi release are related in general, and how Pi is released by actin in particular.