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. It remains unclear how F-actin hydrolyzes ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here, we present cryo-EM structures of F-actin in all nucleotide states, polymerized in the presence of Mg²⁺ or Ca²⁺, at resolutions (∼2.2 Å) that allow for the visualization of hundreds of water molecules. The structures reveal that the G- to F-actin transition 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 the F-actin conformation that allows for 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 waters in the nucleotide binding pocket explain why Ca²⁺-actin exhibits 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.