Actin depolymerizing factor (ADF) and cofilin accelerate actin dynamics by severing and disassembling actin filaments. Here, we present the 3.8 Å resolution cryo-EM structure of cofilactin (cofilin-decorated actin filament). The actin subunit structure of cofilactin (C-form) is distinct from those of F-actin (F-form) and monomeric actin (G-form). During the transition between these three conformations, the inner domain of actin (subdomains 3 and 4) and the majority of subdomain 1 move as two separate rigid bodies. The cofilin–actin interface consists of three distinct parts. Based on the rigid body movements of actin and the three cofilin–actin interfaces, we propose models for the cooperative binding of cofilin to actin, preferential binding of cofilin to ADP-bound actin filaments and cofilin-mediated severing of actin filaments.

The actin filament has clear polarity where one end, the pointed end, has a much slower polymerization and depolymerization rate than the other end, the barbed end. This intrinsic difference of the ends significantly affects all actin dynamics in the cell, which has central roles in a wide spectrum of cellular functions. The detailed mechanism underlying this difference has remained elusive, because high‐resolution structures of the filament ends have not been available. Here, we present the structure of the actin filament pointed end obtained using a single particle analysis of cryo‐electron micrographs. We determined that the terminal pointed end subunit is tilted towards the penultimate subunit, allowing specific and extra loop‐to‐loop inter‐strand contacts between the two end subunits, which is not possible in other parts of the filament. These specific contacts prevent the end subunit from dissociating. For elongation, the loop‐to‐loop contacts also inhibit the incorporation of another actin monomer at the pointed end. These observations are likely to account for the less dynamic pointed end.

Significance: It remains obscure how actin polymerizing and depolymerizing activities cooperate to control diverse actin dynamics. Formins rotate along the long-pitch helix of F-actin during processive actin elongation (helical rotation), which may twist F-actin in the opposite direction of the cofilin-induced twisting. In this study, we show that a mammalian formin mDia1 generates F-actin resistant to cofilin. Tethered F-actin elongating from immobilized mDia1 contained a less twisted portion in EM analysis and exhibited resistance to the severing activity of cofilin. In cells, overexpression of an active mDia1 mutant, which harbors N-terminal regulatory domains, prolonged F-actin lifetime and accelerated dissociation of cofilin. Helical rotation of formins may thus facilitate the formation of stabilized F-actin resistant to actin severing activities of cofilin.

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–PO ₃ ⁻ 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.

Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5′-triphosphate (GTP)–bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the “+” end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.