Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains

Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the a ffinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + P-i using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in E scherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMPPNP. Ye t, in the presence of ADP + P-i, there is a decrease in the affinity of bot h MC1 and MC6 for microtubules, The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent K-d(Pi) for the two phosphate sites at 23.3 and 1.9 parent mM, respectively. The result s indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s(-1 ), yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consi stent with a model for the ATPase cycle in which ATP hydrolysis occurs on t he microtubule followed by detachment as the Ncd.ADP.P-i intermediate.