Figure 3. Kinesin 13 neck obstructs adjacent binding-site access on curved /-heterodimers. (A) Representative class averages for dolastatin rings decorated with M, NM, and FL. Bars, 100 Å. (B) Representative class averages (M, NM, and FL), difference maps (NM-M, FL-M, and FL-NM), and Student’s t test maps (right column) for motor–heterodimer cutouts. Arrowheads mark the adjacent binding site on the curved protofilament, and arrows mark the position of extra density seen in NM and FL. Bars, 50 Å. (C) Model of the crystal structures of truncated NM (Protein Data Bank reference codes 2HEH and 1v8k) fitted onto straight (Protein Data Bank reference code 1tub) and bent tubulin (Protein Data Bank reference code 1sa0). The region in red corresponds to the neck domain, the red arrow indicates the difference in neck orientation between the two crystal struc- tures, the region in yellow indicates the -relay helix, and other important domains are marked. kinesin 13 accommodates the compact structure of NM in adja- cent M densities along a curved protofilament (Tan et al., 2008). Further examination of the fitted crystal structures (Fig. 3 C) show that the C terminus of NM extends parallel to the -4 relay helix (Fig. 3 C, yellow) toward adjacent protofilaments. Exten- sion along this axis of the C-terminal coiled coil of FL kine- sin 13 could prime the motor for dimerization in a manner that allows adjacent protofilament access. Significantly, because ki- nesin 13 dimerizes through both its N and C termini (Maney et al., 2001) and the N terminus would extend from the neck domain, the motor would be locked on both ends in a conformation with better access to adjacent protofilaments than to adjacent sites on the same protofilament. Model for interaction of kinesin 13 with curved protofilaments The monomeric NM construct has reduced ability to bind curved protofilaments (Figs. 2 and 3), suggesting that the neck domain alone is sufficient to preclude adjacent site access; however, NM- induced stack formation (Fig. 1) would argue that this domain in- teracts with adjacent protofilaments as well as precluding access to binding sites on a single protofilament. Previous studies have reported that the NM construct exists in an extended and compact state (Ems-McClung et al., 2007), and the existence of two differ- ent NM x-ray structures (Fig. 3 C) confirms the flexibility of this domain (Ogawa et al., 2004). Importantly, there is precedent for regulation of the neck by the N terminus (Ems-McClung et al., 2007), and our observations that a construct consisting of the N terminus in addition to NM assumes a more compact conforma- tion on dolastatin rings (Fig. S1 C) support this idea. Together with our results, these observations suggest that the neck domain of NM can assume an extended conformation that precludes (Fig. 4 A, top) and a compact conformation that accommodates (Fig. 4 A, bottom) adjacent site binding on curved protofilaments. Our modeling experiments indicate that both conformations of NM should decorate adjacent binding sites on straight protofilaments (Fig. 3 C and Fig. 4 A). The observations that FL has a reduced ability to decorate curved protofilaments (Figs. 2 and 3) suggest that this construct is sterically hindered from adjacent site binding. The absence of well-resolved densities for the N and C termini or the second poly- peptide of FL in EM averages (Fig. 3, A and B) indicates that these domains are flexible. Extensive conformational variability has been previously reported for FL (Ems-McClung et al., 2007). Together with ring stack formation by FL (Fig. 1), these observa- tions suggest that the second polypeptide of dimeric FL is available for binding to adjacent protofilaments. We propose that the design and size of FL precludes adjacent site binding on straight and curved protofilaments so that this dimeric motor preferentially binds adjacent protofilaments on the MT lattice (Fig. 4 B). Such a configuration of binding by FL would allow increased depolymer- ization efficiency by enabling the dimeric motor to depolymerize two protofilaments at the same time. An intriguing alternative interpretation of nonadjacent binding by FL is one in which motor densities on nonadjacent heterodimers belong to a single dimeric FL molecule. This might be suggestive of a depolymerization mechanism that combines KINESIN 13 BINDING TO CURVED PROTOFILAMENTS • Mulder et al. 55