ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation1–3. In turn, force4,5 and actin filament (F-actin) nucleotide state6 regulate actin dynamics by tuning F-actin’s engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP–F-actin and ADP-Pi–F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP–F-actin and ADP-Pi–F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation.

ATP hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation. Force and actin filament (F-actin) nucleotide state in turn modulate the engagement of actin binding proteins (ABPs) to regulate actin dynamics through unknown mechanisms. Here, we show that bending forces evoke structural transitions in F-actin which are modulated by actin nucleotide state. Cryo-electron microscopy (cryo-EM) structures of ADP- and ADP-Pi-F-actin with sufficient resolution to visualize bound solvent reveal inter-subunit interactions primarily bridged by waters which could contribute to lattice flexibility. Despite substantial ordered solvent differences in the nucleotide binding cleft, these structures feature essentially indistinguishable protein backbone conformations which are unlikely to be discriminable by ABPs. We next introduce a machine-learning enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain level detail. Bent F-actin structures reveal major rearrangements at inter-subunit interfaces characterized by striking alterations of helical twist and deformations of individual protomers which are distinct in ADP- and ADP-Pi-F-actin. This suggests phosphate rigidifies actin subunits to alter F-actin's bending structural landscape. We therefore propose actin nucleotide state serves as a co-regulator of F-actin mechanical regulation, as bending forces evoke nucleotide-state dependent conformational transitions that could be readily detected by ABPs.

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.

No abstract

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.