Structural basis of actin filament assembly and aging.

The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state^1-3. It remains unclear how F-actin hydrolyses ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here we present cryo-electron microscopy structures of F-actin in all nucleotide states, polymerized in the presence of Mg²⁺ or Ca²⁺ at approximately 2.2 Å resolution. The structures show that actin polymerization induces the relocation of water molecules in the nucleotide-binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (P_i) is closed in all structures, indicating that P_i release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and P_i release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of water molecules in the nucleotide-binding pocket explain why Ca²⁺-actin shows slower polymerization rates than Mg²⁺-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.