The Kinesin-1 Motor Domain is Regulated by a Direct Interaction of its Head and TailPublic Deposited
Kinesin-1 is a motor protein that transports cargo along microtubules. Inside cells, the majority of kinesin-1 is regulated to conserve ATP and ensure its proper intracellular distribution and coordination with other motors. Regulated kinesin-1 is folded in half, and interactions between coiled-coil regions near the N-terminal enzymatically active heads and the C-terminal regulatory tails bring these globular elements in close proximity to stabilize the folded conformation. However, it has remained a mystery how the kinesin-1 tail inhibits ADP release and thus catalytic activity in this folded conformation. To test whether the tail regulates the head by directly interacting with it, my collaborators and I performed photochemical cross-linking experiments on head and tail domains in trans and analyzed these results using mass spectrometry. These techniques provided the first evidence of a direct contact between the head and tail domain and allowed for mapping of the inhibitory interaction; the regulatory QIAKPIRP motif of the tail interacts with Switch I and the nucleotide pocket of the motor domain. Cryo-electron microscopy on a head-tail crosslink confirmed this finding and provided a possible mechanism for regulation, as Switch I was observed for the first time in an "open" position, a conformation with high ADP affinity. A new state for kinesin-1 was also seen, in which the tail simultaneously interacts with Switch I and the microtubule. In this state the motor is regulated through the interaction of the QIAKPIRP motif of the tail with Switch I, but remains microtubule-bound through stabilizing interactions between the tail and tubulin. The physiological relevance of this state remains unknown. Electron paramagnetic resonance and fluorescence assays were used to examine how the tail, specifically the K922 residue, inhibits ADP release. The tail-induced conformational restriction of the nucleotide pocket is distinct from the conformational changes caused by microtubule binding and occurs independent of the regulatory K922 residue. While the exact mechanism of inhibition could not be determined, structural and biochemical homology with G-proteins suggests that the tail may be acting in a manner similar to guanine nucleotide dissociation inhibitors; this mode of regulation may be a common feature among kinesin family members.