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 affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results 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 consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.
Foster
K A
KA
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Correia
J J
JJ
Gilbert
S P
SP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000494:Allosteric Regulation; D019281:Dimerization; D029721:Drosophila Proteins; D017022:Flow Injection Analysis; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008956:Models, Chemical; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014461:Ultracentrifugation
D029721:Drosophila Proteins; D020409:Molecular Motor Proteins; D010710:Phosphates; D011994:Recombinant Proteins; C066696:ncd protein, Drosophila; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins