Li, X.; Hu, J.; Sun, Y.; Xu, C.; Fu, Y.

Actin nucleation is a rate-limiting step in F-actin assembly. However, early actin trimers are intrinsically unstable, with limited inter-subunit contacts rendering them prone to dissociation. VASP is an important regulator of actin polymerization and assembly, but how it stabilizes unstable trimeric actin nucleus remains poorly understood at the molecular level. Here, we use coarse-grained and all-atom molecular dynamics simulations to characterize the interfacial stability and conformational features of VASP-bound actin trimers. Energetic and interfacial analyses show that VASP preferentially anchors the laterally associated subunit through multivalent interactions and reinforces its interfaces with the longitudinal actin dimer. Within the VASP-bound trimer, the three actin subunits retain distinct local conformational features, as reflected by differences in W-loop gating and SD1-to-SD4 dihedral angle. Across replicate simulations, VASP stabilizes the actin nucleus while preserving subunit-specific conformational heterogeneity, rather than uniformly rigidifying the trimer. These findings provide a molecular basis for VASP-mediated stabilization of early actin nuclei and offer broader mechanistic insights into the regulation of protein assembly.