Structural Basis of Kinesin-1 Autoinhibition and Its Control of Microtubule-Based Motility

Abstract

Kinesin-1 was the first identified microtubule-based motor protein that drives anterograde intracellular transport of diverse cargoes in eukaryotic cells. Improper regulation and kinesin-1 defects are implicated in multiple neurological disorders as well as pathogens hijacks kinesin-1 to deliver their cargoes. Despite its importance, the molecular mechanisms governing kinesin-1 regulation and activation remains poorly understood. Here, we report the cryo-EM structure of the autoinhibited kinesin-1 heterotetramer and validate it using crosslinking mass spectrometry. The structure reveals a 36-nm 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 wedge 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 movement. In addition, the structure shows that the KLC C-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, compete 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.