Microtubule association induces a Mg-free apo-like ADP pre-release conformation in kinesin-1 that is unaffected by its autoinhibitory tail

Abstract

Kinesin-1 is a processive dimeric ATP-driven motor that transports vital intracellular cargos along microtubules (MTs). If not engaged in active transport, kinesin-1 limits futile ATP hydrolysis by adopting a compact autoinhibited conformation that involves an interaction between its C-terminal tail and the N-terminal motors domains. Here, using a chimeric kinesin-1 that fuses the N-terminal motor region to the tail and a tail variant unable to interact with the motors, we employed high-resolution cryo-EM in the presence of MTs to investigate elements of the mechanochemical cycle. We describe a missing structure for the proposed two-step allosteric mechanism of ADP release, the ATPase rate limiting step. It shows that MT association induces remodeling of the hydrogen bond network at the nucleotide binding site triggering removal of the Mg2+ ion from the Mg2+-ADP complex resulting in a strong MT-binding apo-like state before ADP dissociation. We further demonstrate that tail association does not directly affect this mechanism, nor the adoption of the ATP hydrolysis-competent conformation, nor neck linker docking/undocking, even when zippering the two motor domains. Based on this structural evidence, we propose a revised mechanism for tail-dependent kinesin-1 autoinhibition and suggest a possible explanation for its characteristic pausing behavior on MTs.