JCB: REPORT Y G O L O I B L L E C F O L A N R U O J E H T A new model for binding of kinesin 13 to cur ved microtubule protofilaments Anke M. Mulder,1 Alex Glavis­ Bloom,1 Carolyn A. Moores,1,2 Michael Wagenbach,3 Bridget Carragher,1 Linda Wordeman,3 and Ronald A. Milligan1 1Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037 2School of Crystallography, Birbeck College, University of London, London WC1E 7HX, England, UK 3Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98104 K hydrolysis to do work on microtubules (MTs). Most inesin motor proteins use adenosine triphosphate kinesins walk along the MT, but class 13 kinesins instead uniquely recognize MT ends and depolymerize MT protofilaments. We have used electron microscopy (EM) to understand the molecular interactions by which kinesin 13 performs these tasks. Although a construct of only the motor domain of kinesin 13 binds to every hetero­ dimer of a tubulin ring, a construct containing the neck and the motor domain occupies alternate binding sites. Likewise, EM maps of the dimeric full­ length (FL) protein exhibit alternate site binding but reveal density for only one of two motor heads. These results indicate that the second head of dimeric kinesin 13 does not have access to adjacent binding sites on the curved proto­ filament and suggest that the neck alone is sufficient to obstruct access. Additionally, the FL construct promotes increased stacking of rings compared with other con­ structs. Together, these data suggest a model for kinesin 13 depolymerization in which increased efficiency is achieved by binding of one kinesin 13 molecule to adja­ cent protofilaments. Introduction Microtubules (MTs) are a vital part of the cellular cytoskeleton and are intimately involved in processes such as the transport of molecular cargo, proper chromosome attachment during cell division, and cell structure and morphology (Nogales, 2001; Heald and Nogales, 2002). MTs are dynamic polymers built from heterodimers of /-tubulin whose GTP-bound state has a straight conformation that lends itself to polymer- ization and whose GDP-bound state has a bent conformation that encourages depolymerization of the filament (Desai and Mitchison, 1997). The ability to polymerize and depolymerize in accordance with the localized needs of the cell is an impor- tant aspect of MT function and is tightly controlled by a vari- ety of MT-associated proteins (Walczak, 2000). Among these regulators of MT growth and shrinkage are depolymerizing motor proteins such as kinesin 13, which actively depolymer- ize the MT filament using energy derived from ATP hydrolysis (Walczak et al., 1996; Maney et al., 1998; Desai et al., 1999; Moores et al., 2002). In contrast to conventional kinesins, which walk along an MT track, kinesin 13s do not walk but instead uniquely recog- Correspondence to Ronald A. Milligan: milligan@scripps.edu Abbreviations used in this paper: FL, full length; MCAK, mitotic centromere- associated kinesin; MT, microtubule; NM, neck plus motor core. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 185 No. 1 51–57 www.jcb.org/cgi/doi/10.1083/jcb.200812052 nize MT ends and depolymerize MT protofilaments. It was originally thought that the internal sequence location of the kinesin 13 catalytic domain contributed to its depolymerization activity, as the plus or minus end directionality of other kinesins correlates with N- or C-terminal localizations of the catalytic domain (Vale and Fletterick, 1997; Miki et al., 2001; Lawrence et al., 2004). However, studies have shown that the kinesin 13 catalytic domain alone is sufficient for depolymerization of MT filaments (Moores et al., 2002; Niederstrasser et al., 2002) so that the unique activity of kinesin 13 must be contained within the sequence of its motor core. Structural analysis of the kinesin 13 motor core has revealed that the MT-binding face of the pro- tein has a convex conformation that is strikingly complemen- tary to the surface of a bent protofilament (Ogawa et al., 2004; Shipley et al., 2004), suggesting that depolymerization activity stems, at least in part, from unique interactions between the convex shape of the kinesin 13 motor core and bent tubulin polymer (Ogawa et al., 2004; Shipley et al., 2004). Suggestively, tubulin flexibility is an important stimulator of kinesin 13 ATP © 2009 Mulder et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publica- tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). JCB 51