Abstract

Molecular motors convert stochastic ATP chemistry into directional motion. Modern nonequilibrium theories explain this behavior through kinetic asymmetry, yet the molecular mechanisms that generate kinetic asymmetry in AAA+ unfoldases remain unresolved. Cryo-electron microscopy has consistently revealed unfoldases take on an asymmetric helical staircase structure, inspiring the proposed processive hand-over-hand translocation mechanism. In contrast, biochemical and single-molecule studies have reported bursts, slips, and variable step sizes indicative of stochastic behavior. How these observations are reconciled and how directional motion emerges from stochastic ATP hydrolysis remain central questions. Here we use all-atom molecular dynamics simulations of the AAA+ unfoldases Yme1 and Vps4 to explore structural changes induced by hydrolysis, ADP release, and ATP association. The simulations reveal coexisting pre- and post-hydrolysis conformations consistent with experimental cryo-EM densities while also uncovering interconnected allosteric communication networks. One network links the Walker B glutamate to the proximal subunit's nucleotide binding pocket (NBP), providing a mechanism for backward propagation of hydrolysis competence around the hexamer. Another network couples NBP and salt bridge rearrangements to pore-loop dynamics, connecting subunit position rather than ATP hydrolysis to substrate release. Simulations of nucleotide exchange intermediates further show that ATP binding before ADP and/or Mg2+ release can induce transient strain leading to alternative hydrolysis-competent configurations that still favor forward progression, providing a structural basis for some of the experimentally observed stochastic behavior. Together, these findings suggest that structural asymmetry creates position-dependent allosteric interactions that generate kinetic asymmetry, biasing a fundamentally stochastic ATPase cycle toward directional substrate translocation while still permitting variation in stepping behavior.