Structural basis of mechano-chemical coupling by the mitotic kinesin KIF14

Abstract

KIF14 is a mitotic kinesin protein important for cytokinesis. Its overexpression is associated with a variety of cancers and mutations in KIF14 result in cerebral and renal development defects.Like other kinesins, KIF14 contains a highly conserved catalytic motor domain where the energy from ATP hydrolysis is converted to directed movement along microtubules. Although much is known regarding the molecular mechanism of kinesin motility, there is a lack of structural information of kinesin-microtubule interactions at sufficient resolution to unambiguously assess how conformational changes related to ATP hydrolysis, microtubule binding and translocation are coupled. Here we determined the near-atomic resolution cryo-electron microscopy structures of five different KIF14 constructs bound to microtubules in the presence of different nucleotide analogues mimicking distinct steps of the ATPase cycle. Eighteen independent structures together with supporting functional assays provide a comprehensive view of the kinesin conformational changes occurring with microtubule and nucleotide binding. Our data shows that: 1) microtubule binding induces opening of the KIF14 nucleotide binding pocket; 2) AMP-PNP and ADP-AlF x induce closure of the nucleotide binding pocket in microtubule bound KIF14 and this conformational change is allosterically controlled by the neck-linker domain; 3) the necklinker domain when undocked prevents the nucleotide-binding-pocket to fully close and dampens ATP hydrolysis; 4) fifteen neck-linker residues are required to assume the docked conformation; 5) the nucleotide analogue ADP-AlF x adopts a distinct configuration in an open nucleotide-binding-pocket; 6) the neck-linker position controls the hydrolysis step rather than 2 nucleotide binding in the KIF14 ATPase cycle; 7) the two motor domains of a KIF14 dimer adopt distinct conformations when simultaneously bound to the microtubule. These observations provide the structural basis for a coordinated chemo-mechanical kinesin plus end translocation model.

Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape

“…The processing was done as previously described 13 . Movie frames were aligned with motioncor2 53 generating both doseweighted and non-dose-weighted sums and correcting for magnification anisotropy (Supplementary Table 1).…”

“…Helical reconstruction on 15R microtubules was performed using a helical-single-particle 3D analysis workflow in Frealign 56 , as described previously 13,16 , with each half filament contributing to a distinct half dataset. The box size used are indicated in Supplementary Table 1.…”

“…To select for tubulins bound to kinesin motors and to improve the resolution of the kinesin-tubulin complexes the procedure HSARC 13 was used for these one-head-bound states. The procedure follows these steps:…”

“…First, atomic models for the kinesin chains were generated from their amino-acid sequence by homology modeling using Modeller 61 . The initial tubulin structures were taken from 13 . Second, the protein chains were manually placed into the cryo-EM densities and fitted as rigid bodies using UCSF-Chimera 62 .…”

“…At this time, our knowledge of the catalytic cycles of kinesins is limited to motile kinesins that are incapable of microtubule depolymerization, and to microtubule-depolymerizing kinesins that are non-motile. In motile kinesins, such as kinesin-1 and kinesin-3, microtubule lattice binding by one of their two motor domains opens the nucleotide-binding pocket, allowing ADP release [12][13][14] . Subsequent entry of ATP initiates closing of the nucleotide-binding pocket and docking of a short peptide, known as the neck-linker, along the side of the motor domain.…”