Kinesin-1 utilizes a hierarchical folding mechanism to promote autoinhibition

The spatiotemporal regulation of organelle positioning by kinesin motor proteins is critical for proper cellular function. Conventional kinesin-1 (kinesin) is the primary anterograde motor in cells. Kinesin is composed of heavy and light chains, existing as a homodimer of heavy chains and heterotetramers of heavy and light chains. While there is a consensus that autoinhibited kinesin adopts a folded conformation, the molecular basis for kinesin autoinhibition remains unknown. Here, we combined cross-linking mass spectrometry (XL-MS) and electron microscopy (EM) to determine the architecture of the full-length autoinhibited kinesin. First, we utilized structural studies with negative stain electron microscopy to characterize the folded conformation of kinesin homodimers and heterotetramers. Both biochemical and structural analysis showed that kinesin exists in folded and compact conformations. To determine the folding pattern of kinesin, we combined protein structure prediction using AlphaFold and XL-MS to derive a molecular model of the auto-inhibited full-length kinesin. Surprisingly, we discovered that the originally proposed ‘hinge’ was not the true hinge. Instead, a break in coiled-coiled 3 allows four coiled-coil domains to fold the kinesin. XL-MS analysis of the heterotetramer showed that kinesin light chains do not change the folding pattern, suggesting that light chains stabilize the inhibited state, not stabilizing a new structural state. We validated and extended our structural analysis of kinesin autoinhibition via in vivo and in vitro mutagenesis studies that showed that disruption of multiple regions is required to fully activate the full-length kinesin motor. Importantly, we show that autoinhibition requires many more interactions than the inhibitory tail peptide, providing the first complete view of autoinhibition. Our structural description offers a conceptual framework for understanding how activation factors such as MAP7 and cargo relieve autoinhibition.