Structural basis of kinesin-1 autoinhibition and its control of microtubule-based motility.

Kinesin-1 was the first microtubule motor identified, responsible for anterograde transport of diverse cargo in eukaryotic cells. Defects or misregulation of kinesin-1 is linked to multiple neurological disorders, and various pathogens exploit kinesin-1 to transport their cargo. In the absence of cargo, kinesin-1 adopts a compact autoinhibited conformation to enable its spatiotemporal regulation and prevent futile energy consumption. Despite its importance, the structural mechanisms for kinesin-1 autoinhibition and activation remain poorly understood. Here, we report the cryo-electron microscopy structure of the autoinhibited kinesin-1 heterotetramer and validate it using cross-linking mass spectrometry. The structure reveals a 36-nanometer particle in which the kinesin heavy chains (KHCs) adopt a head-to-tail configuration, stabilized by asymmetrically arranged kinesin light chain (KLC) tetratricopeptide repeat (TPR) domains that bind across folded KHC coiled coils and in between the KHC motor domains. This architecture inhibits kinesin motility by constraining the dimeric motor domains in a configuration that is incompatible with processive motility. In addition, the structure shows that the KLC carboxyl-terminal helices occlude the TPR cargo-binding interfaces, revealing a second layer of autoinhibition that directly blocks cargo engagement. Functional studies and structural modeling suggest that binding of regulatory factors, such as MAP7D3, competes with intramolecular KHC coiled-coil interactions, resulting in the unfurling of the autoinhibited structure and activating motor motility. These findings provide a molecular framework for understanding kinesin-1 regulation and its implications for intracellular transport.