Abstract
Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in solution are not equivalent; ADP is held more tightly at one site than at the other. Upon microtubule binding, fast release of ADP from the first motor domain is stimulated at 18 s(-1), yet rate-limiting ADP release from the second motor domain occurs at 1.4 s(-1). We propose that the head with the low affinity for ADP binds the microtubule first to establish the directional bias of the microtubule.Ncd intermediate where one motor domain is bound to the microtubule with the second head detached and directed toward the minus-end of the microtubule. The force generating cycle is initiated as ATP binds to the empty site of the microtubule-bound head. ATP hydrolysis at head 1 is required for head 2 to bind to the microtubule. The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor.
Highlights
Similarity, there are striking differences in their motor behavior that are important for biological function in vivo
We assumed that the detached motor domain would bind mant-ADP more tightly than the motor domain bound to the microtubule because microtubules activate ADP release from 0.005 sϪ1 in the absence of microtubules to ϳ2 sϪ1 at high microtubule concentrations (14, 15, 30 –34, 37)
The protein concentration determined by the Bio-Rad protein assay was comparable to the concentration of active sites determined by acid-quench burst experiments and the creatine kinase active site titration [15, 34]. These results indicate that one motor domain binds ADP tightly while the other head binds ADP more weakly, resulting in a rapid equilibrium at this weak site. These data suggest that upon dimerization, an asymmetry is established between the motor domains, and this asymmetry is intrinsic to the dimer before it interacts with the microtubule
Summary
Vol 276, No 22, Issue of June 1, pp. 19259 –19266, 2001 Printed in U.S.A. Received for publication, September 12, 2000, and in revised form, January 19, 2001 Published, JBC Papers in Press, March 2, 2001, DOI 101.074/jbc.M008347200. Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor. Ncd is a microtubule-activated ATPase of the kinesin superfamily involved in spindle assembly and stabilization during female meiosis and the early divisions of the Drosophila embryo [1,2,3,4]. The motor domains of Ncd and kinesin are ϳ40% identical in amino acid sequence, and their three-dimensional atomic structures are nearly superimposable (8 –10).
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