Equilibrium Binding Studies of Non-claret Disjunctional Protein (Ncd) Reveal Cooperative Interactions between the Motor Domains

doi:10.1074/jbc.273.52.35307

Equilibrium Binding Studies of Non-claret Disjunctional Protein (Ncd) Reveal Cooperative Interactions between the Motor Domains*

From the ^‡Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 and the ^¶Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216

Abstract

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-β,γ-imidodiphosphate (AMP-PNP), ADP, or ADP + P_i 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 + P_i, 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 apparentK _{d(P_i} ₎ 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·P_i intermediate.

Footnotes

↵* This work was supported by Grant GM 54141 (to S. P. G.) from the National Institutes of Health, IRG-58-35 from the American Cancer Society, and in part by a Basil O’Connor Starter Scholar Research Award 5-FY95-1136 from the March of Dimes Birth Defects Foundation. This is University of Mississippi Medical Center Analytical Ultracentrifuge Facility publication number 19.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ University of Pittsburgh Chancellor’s Scholar and the recipient of a Beckman Scholarship from the Arnold and Mabel Beckman Foundation and a Barry M. Goldwater Scholarship in support of undergraduate research.
↵‖ Recipient of an American Cancer Society Junior Faculty Research Award JFRA-618. To whom correspondence should be addressed: Dept. of Biological Sciences, 518 Langley Hall, University of Pittsburgh, Pittsburgh, PA 15260. Tel.: 412-624-5842; Fax: 412-624-4759; E-mail: spg1+{at}pitt.edu.
↵2 C. C. Wadsworth and S. P. Gilbert, personal communication.
Abbreviations:

Ncd

non-claret disjunction

MC1

Ncd construct of Leu²⁰⁹–Lys⁷⁰⁰ amino acid residues

MC6

Ncd construct of Met³³³–Lys⁷⁰⁰ amino acid residues

AMP-PNP

5′-adenylyl-β,γ-imidodiphosphate

ATPγS

adenosine 5′-O-(3-thiotriphosphate)

PIPES

1,4-piperazinediethanesulfonic acid

Mt·N

microtubule·Ncd complex

a.a.

amino acids

Mgacetate

magnesium acetate

Kacetate

potassium acetate

DTT

dithiothreitol.
- Received August 26, 1998.
- Revision received October 9, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.