Figure 2. M, NM, and FL binding to dolastatin rings. (A) Representative SDS-PAGE gel of sedimentation assays conducted with M, NM, and FL constructs incu- bated with dolastatin rings in the AMPPNP state. The molar concentrations are 5.1, 3.6, 5.1, and 2.0 µM for M, NM, FL, and heterodimeric tubulin (tub). s, supernatant; p, pellet; MW, molecular weight. (B) OD measurements with a BSA standard curve using Im- ageJ (Abramoff et al., 2004) software allowed calcu- lation of the motor to heterodimer ratio normalized for starting motor concentration. Values were determined from duplicate experimental runs (not depicted), and repeated sedimentation assays resulted in a binding trend of M > NM > FL for dolastatin rings. Graph dis- plays mean ± SEM. *, P < 0.05; ***, P < 0.001. site binding (Fig. S1 C), confirming that dimeric kinesin 13 is precluded from adjacent site binding on the curved protofila- ment and suggesting that the N terminus is not involved in the preclusion of adjacent site binding. These observations, together with stack formation data (Fig. 1), suggest that FL would rather bind nearby protofilaments than occupy adjacent binding sites on a single curved protofilament. Interaction of M, NM, and FL with bent /-tubulin heterodimer To locate the additional domains present in the FL and NM con- structs and better understand their contribution to alternate site binding, we computationally cut out individual motor–heterodimer complexes from the 14-mer ring class averages and analyzed these particles by single particle averaging methods (Fig. 3 B). Visual comparison of motor–heterodimer averages revealed key differences in the appearance of the M, NM, and FL motor den- sities. The M construct assumed a compact conformation on the heterodimer compared with more extended conformations as- sumed by the NM and FL constructs (Fig. 3 B, M vs. NM and FL). The NM and FL constructs had the characteristic arrow- head shape that has been previously observed for MCAK (Ogawa et al., 2004; Shipley et al., 2004). The larger FL con- struct revealed some diffuse density to either side of the motor core (Fig. 3 B, FL), likely as a result of conformational hetero- geneity in its additional N- and C-terminal domains. Difference map comparisons between motor–heterodimer averages of M, NM, and FL confirmed the statistically significant absence of motors to either side of the NM and FL cores (Fig. 3 B, arrowheads, P < 0.01), a feature that was notably absent in the FL- NM difference map (Fig. 3 B, bottom). Additionally, FL-M and FL-NM difference maps did not visualize a density attributable to the second polypeptide of the dimeric FL construct (Fig. 3 B, mid- dle and bottom), suggesting that it does not assume a fixed position on the ring and that this density was lost during averaging of multi- ple images. These results support our findings that NM and FL en- gage in alternate site binding on the curved protofilament and that the two heads of dimeric FL are not engaged on the same ring. Importantly, NM-M and FL-M difference maps revealed additional density to the minus end side of the NM and FL mo- tor cores (Fig. 3 B, arrows, P < 0.01), a feature that was absent in the FL-NM difference map (Fig. 3 B, bottom). The additional density in the NM and FL averages was observed for motor– heterodimers excised from multiple class averages and was not observed within construct datasets. That this density was ob- served in both the FL-M and NM-M difference maps but not in the FL-NM difference map suggests that the extended confor- mations of FL and NM as compared with M are at least in part attributable to the neck domain. Models of a kinesin 13 NM crystal structure fitted onto straight and bent protofilaments reveal significant steric hindrance to adjacent site binding on the curved protofilament (Fig. 3 B, top vs. bottom; Nogales et al., 1999; Ogawa et al., 2004; Ravelli et al., 2004). The NM crystal structure indicates that such steric hindrance could be caused by the neck domain alone (Fig. 3 C, red domain) or through interaction of the neck with the L2 loop. No- tably, a second crystal structure of NM (Ogawa et al., 2004) has a more compact conformation in which the neck domain points to- ward the adjacent protofilament (Fig. 3 C, red arrow); such a con- formation of NM could potentially accommodate adjacent site binding on the curved protofilament. Indeed, a recent structure of bracelet rings induced by M from Drosophila melanogaster 54 JCB • VOLUME 185 • NUMBER 1 • 2009