The Kinesin-1 tail conformationally restricts the nucleotide pocket.

We have used electron paramagnetic resonance and fluorescence spectroscopy to study the interaction between the kinesin-1 head and its regulatory tail domain. The interaction between the tails and the enzymatically active heads has been shown to inhibit intrinsic and microtubule-stimulated ADP release. Here, we demonstrate that the probe mobility of two different spin-labeled nucleotide analogs in the kinesin-1 nucleotide pocket is restricted upon binding of the tail domain to kinesin-1 heads. This conformational restriction is distinct from the microtubule-induced changes in the nucleotide pocket. Unlike myosin V, this tail-induced restriction occurs independent of nucleotide state. We find that the head-tail interaction that causes the restriction only weakly stabilizes Mg(2+) in the nucleotide pocket. The conformational restriction also occurs when a tail construct containing a K922A point mutation is used. This mutation eliminates the tail's ability to inhibit ADP release, indicating that the tail does not inhibit nucleotide ejection from the pocket by simple steric hindrance. Together, our data suggest that the observed head-tail interaction serves as a scaffold to position K922 to exert its inhibitory effect, possibly by interacting with the nucleotide alpha/beta-phosphates in a manner analogous to the arginine finger regulators of some G proteins.

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail.

Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-A cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.