Abstract
Kinesin-1 is the first identified microtubule-based motor protein that drives anterograde intracellular transport of diverse cargoes in eukaryotic cells. Defects in kinesin-1 activity have been implicated in various human neurological disorders and intracellular pathogens such as bacteria and viruses hijack its activity. Despite its importance, the molecular mechanisms governing kinesin-1 activation remains poorly understood. Here, we report an 8.0-Å cryo-electron microscopy structure of the kinesin-1 heterotetramer composed of two kinesin heavy chains (KHC) and two light chains (KLC), in its autoinhibited conformation. Our structure reveals a complex 36-nm head-to-tail assembly in which self-folded dimeric KHCs are stabilized by asymmetrically arranged KLCs through a combination of intramolecular and intermolecular interactions. This state inhibits kinesin motility by constraining the dimeric motor domains in a configuration that is incompatible with motility. Notably, our structure also shows that the dimeric KLC TPR cargo-binding domains are occluded, providing a structural basis for the autoinhibition of both motor activity and cargo binding. Functional studies along with structural modeling suggest that binding of known regulatory factors, such as MAP7D3, to the KHC coiled coils likely competes with intramolecular coiled-coil interactions, resulting in the unfurling of the autoinhibited structure and activation of motor activity. Our findings provide a molecular framework for understanding the regulation of kinesin-1 activity and its implications for intracellular transport.