To elucidate the structural basis of the mechanism of microtubule depolymerization by kinesin-13s, we analyzed complexes of tubulin and the Drosophila melanogaster kinesin-13 KLP10A by electron microscopy (EM) and fluorescence polarization microscopy. We report a nanometer-resolution (1.1 nm) cryo-EM three-dimensional structure of the KLP10A head domain (KLP10AHD) bound to curved tubulin. We found that binding of KLP10AHD induces a distinct tubulin configuration with displacement (shear) between tubulin subunits in addition to curvature. In this configuration, the kinesin-binding site differs from that in straight tubulin, providing an explanation for the distinct interaction modes of kinesin-13s with the microtubule lattice or its ends. The KLP10AHD-tubulin interface comprises three areas of interaction, suggesting a crossbow-type tubulin-bending mechanism. These areas include the kinesin-13 family conserved KVD residues, and as predicted from the crossbow model, mutating these residues changes the orientation and mobility of KLP10AHDs interacting with the microtubule.

To investigate the mechanism of kinesin13-induced microtubule depolymerization, we have calculated a three-dimensional (3D) map of the kinesin13-microtubule ring complex, using cryoelectron microscopy (cryo-EM) and image analysis. An atomic model of the complex was produced by docking the crystal structures of tubulin and a kinesin13 motor domain (MD) into the 3D map. The model reveals a snapshot of the depolymerization mechanism by providing a 3D view of the complex formed between the kinesin13 MD and a curved tubulin protofilament (pf). It suggests that contacts mediated by kinesin13 class-specific residues in the putative microtubulebinding site stabilize intra-dimer tubulin curvature. In addition, a tubulin-binding site on the kinesin13 MD was identified. Mutations at this class-conserved site selectively disrupt the formation of microtubule-associated ring complexes. ACCESSION NUMBERSThe electron density map has been deposited in the EM Data Bank (EMDB) under accession number EMD-5027, and the atomic model of the complex has been deposited in the Protein Data Bank under PDB code 3EDL. SUPPLEMENTAL DATA Supplemental Data include two figures and can be found with this article online at

Kinesins-13s are members of the kinesin superfamily of motor proteins that depolymerize microtubules (MTs) and have no motile activity. Instead of generating unidirectional movement over the MT lattice, like most other kinesins, kinesins-13s undergo one-dimensional diffusion (ODD) and induce depolymerization at the MT ends. To understand the mechanism of ODD and the origin of the distinct kinesin-13 functionality, we used ensemble and single-molecule fluorescence polarization microscopy to analyze the behavior and conformation of Drosophila melanogaster kinesin-13 KLP10A protein constructs bound to the MT lattice. We found that KLP10A interacts with the MT in two coexisting modes: one in which the motor domain binds with a specific orientation to the MT lattice and another where the motor domain is very mobile and able to undergo ODD. By comparing the orientation and dynamic behavior of mutated and deletion constructs we conclude that 1) the Kinesin-13 class specific neck domain and loop-2 help orienting the motor domain relative to the MT. 2) During ODD the KLP10A motor-domain changes orientation rapidly (rocks or tumbles). 3) The motor domain alone is capable of undergoing ODD. 4) A second tubulin binding site in the KLP10A motor domain is not critical for ODD. 5) The neck domain is not the element preventing KLP10A from binding to the MT lattice like motile kinesins.

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