Ka. Foster et al., Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains, J BIOL CHEM, 273(52), 1998, pp. 35307-35318
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.