LS-5-IN-1734 Mutual Conformational Changes of Kinesin (KIF5) and GTP-Microtubule upon their Binding and the Mechanism of the Directional Transport revealed by the Cryoelectron Microscopy and Super-resolution Microscopy (PALM).

Intracellular transport is fundamental for cellular morphogenesis and functions not only in polarized neurons but also cells in general. We identified kinesin superfamily molecular motors (KIFs) and have been studying the mechanism of intracellular transport including the directional transport towards the axon vs dendrites. As a major mechanism for the directional transport we uncovered that the motor domain of KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively move towards the axon. Further, we found that axonal microtubules were preferentially stained by the anti GTP-tubulin antibody(hMB11). Super-resolution microscopy (PALM) combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to GTP- microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GTP- microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.
Microtubule dynamics are regulated by GTP hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the GDP state. As a next step we solved the cryo-EM structure of GTP- microtubule at8.8-A resolution by developing a novel cryo-EM image reconstruction algorithm. Significant changes were detected between GTP- and GDP- microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule binding proteins, such as EB1 or kinesin.
Furthermore , recently we　successfully revealed the 8Å cryo-electron microscopy structure of nucleotide-free KIF5 complexed with GTP-microtubule and the crystal structure of nucleotide-free KIF5 without bound microtubule. These structures illustrated mutual conformational changes induced by the binding of GTP-microtubule and KIF5. Conformational change of tubulin also strengthens the longitudinal contacts of GTP-microtubule mainly from the plus-end side. This could provide the structural keys to solve the molecular mechanisms of preferential binding of KIF5 to GTP-microtubule and cooperative binding of KIF5 to the microtubule.