Thus, our structural and sequence analyses provide an explanation for kinesin13s that form microtubule-associated rings and those that cannot

Thus, our structural and sequence analyses provide an explanation for kinesin13s that form microtubule-associated rings and those that cannot. There is considerable interest in proteins and protein complexes that encircle microtubules, because they may serve as molecular sleeves to keep cargoes attached to the end of dynamic microtubules (Salmon, 2005). the presence of a highly conserved (~320 residues) catalytic or motor domain (MD) that contains the ATP and microtubule-binding sites (Goldstein and Philp, 1999). Most kinesins pull cargoes along microtubules, but an important exception is found in the kinesin13 family, also known as Kin-Is or M kinesins (because of the central location of the MD in the polypeptide chain). Instead of walking along microtubules, kinesin13s promote the disassembly of microtubule ends (Desai et al., 1999), an activity that is important for modulating microtubule dynamics during mitosis (Ganem and Compton, 2004; Rogers et al., 2004) and interphase (Mennella et al., 2005). Kinesin13 activity has been shown to be necessary for proper development of the nervous system (Homma et al., 2003), and its overexpression is associated with some forms of cancer (Ishikawa et al., 2008; Ovechkina and Wordeman, 2003). The kinesin13-induced depolymerization mechanism most likely involves the bending of protofilaments (pfs) at the end of the microtubule (Wordeman, 2005). The bent pfs, which are unable to form lateral contacts, peel off from the microtubule and ultimately cause depolymerization. The kinesin13 MD alone binds to curved tubulin pfs and is sufficient to induce microtubule depolymerization (Moores et al., 2002). A region N-terminal to the MD (~30 residues) called the neck increases depolymerization efficiency (Maney et al., 2001). The physical mechanism by which kinesin13 stabilizes tubulin curvature is unclear. It is also unclear how the highly conserved kinesin MD is adapted in different families, for either walking along or bending tubulin. Constructs containing the kinesin13 MD form oligomeric rings and spirals around microtubules in vitro in the presence of the nonhydrolyzable ATP analog AMP-PNP (Moores Exatecan Mesylate et al., 2006; Tan et al., 2006). No other kinesins have been reported to form such rings. Whether the formation of these oligomers is important for the depolymerization mechanism or some other kinesin13 functionality is still not clear (Davis and Wordeman, 2007; Tan et al., 2006). Image analysis of negatively stained kinesin13-microtubule ring specimens indicated that part of the ring structure was formed by kinesin13 MDs interacting with a curved tubulin pf (Tan Exatecan Mesylate et al., 2006). Thus, these rings/spirals provide a snapshot of the mechanism of kinesin13-induced tubulin curvature. Here, we have used cryo-electron microscopy, 3D reconstruction, and molecular docking to produce an atomic model Exatecan Mesylate of the kinesin13-microtubule ring complex. We then tested the effect, on kinesin13 function, of mutations on residues predicted by the model to from intermolecular contacts. We found a novel microtubule-binding site on the MD that is conserved in the kinesin13 family. The presence of this conserved binding site suggests that the interactions leading to the formation of rings and spirals are relevant to kinesin13 function. == RESULTS == == Ring Formation Is Conserved within the Kinesin13 Family == We previously found that constructs containing the MD of three different kinesin13s from two animal species, Drosophila melanogasterKLP10A and KLP59C andCricetulus griseus(Chinese hamster) MCAK, all form rings around MTs in vitro in the presence of AMP-PNP or ATPS(Tan et al., 2006). To further investigate the generality of this ring-forming ability within the kinesin13 family, we extended our studies to two human kinesin13s. After incubating MD constructs of human KIF2A and KIF2C with microtubules in the presence of AMP-PNP, we observed rings and spirals on microtubules very similar to those formed by other kinesin13s (Figure 1). Exatecan Mesylate == Figure 1 . Human Kinesin13s KIF2A and KIF2C MD Form Rings Around Microtubules in the Presence of AMP-PNP. == (A)Homo sapiensKIF2A (B)H. sapiensKIF2C (C)D. melanogasterKLP10A. Scale bar is 50 nm. These results indicate that the ability to form microtubule-associated rings is conserved within Exatecan Mesylate the kinesin13 family. Only the MD is required to form the rings, so class-conserved residues (present in the kinesin13 family but different in other kinesins that do not form rings) in the MD must mediate the interactions leading to ring formation. To determine candidate residues mediating the formation of the rings, we built an atomic model of the ring/spiral complex based on cryo-EM imaging, followed by molecular docking. == Structure of the KLP10A-Microtubule Ring Complex == Very regular spirals, suitable for 3D reconstruction, form when incubating KLP10A MD constructs with microtubules in the presence of AMP-PNP. For structural analysis, we Mouse monoclonal to CD64.CT101 reacts with high affinity receptor for IgG (FcyRI), a 75 kDa type 1 trasmembrane glycoprotein. CD64 is expressed on monocytes and macrophages but not on lymphocytes or resting granulocytes. CD64 play a role in phagocytosis, and dependent cellular cytotoxicity ( ADCC). It also participates in cytokine and superoxide release selected microtubules with 15 pfs that have no lattice seams (Sosa and Milligan, 1996). Previously we obtained a 3D reconstruction from a negatively stained specimen that allowed us to propose a molecular model of the kinesin13-microtubule ring complex (Tan et al., 2006). However , we noted that in the previous.