Abstract 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 (for example Kif3A and Kif3B in humans) 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 β-hairpin motif) that, in conjunction with the adjacent coiled coil, controls kinesin-2 motility by sequestering the motor domains away from their microtubule track. Our data reveal how Kap3 binds via a multipartite interface with Kif3A and Kif3B. Rather than activating motility directly, Kap3 provides a platform on which cargo adaptors can engage and occlude the β-hairpin motif. Together, these data articulate a structural framework for kinesin-2 activation, recycling by dynein and adaptation for different biological functions.
Webb et al. (Tue,) studied this question.