Beta-hairpin Mechanism of Autoinhibition and Activation in the Kinesin-2 Family

Members of the kinesin-2 family coordinate with other motors to power diverse physiological processes, but the structural mechanisms regulating kinesin-2 activity have been unknown. Distinctively, kinesin-2s canonically function as heterotrimers of two different motor subunits (e.g. Kif3A and Kif3B) and Kap3, but the role of heterotrimerization has yet to fully emerge. Here, we combine structural, cell biological, and single-molecule approaches to dissect kinesin-2 regulation as a heterodimer, heterotrimer, and quaternary complex with a cargo adaptor (APC). We identify a conserved motif in the tail of kinesin-2s (the beta-hairpin motif) that controls kinesin-2 motility by sequestering the motor domains away from their microtubule track, using striking molecular mimicry. Our data reveal how Kap3 binds via a multipartite interface with Kif3A and Kif3B, and - rather than activating motility directly - provides a platform on which cargo adaptors can engage and occlude the beta-hairpin motif. Together, these data articulate a structural framework for kinesin-2 activation, coordination with dynein, and adaptation for different biological functions.