A mechanistic model for Ncd directionality.
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.
Foster
K A
KA
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Mackey
A T
AT
Gilbert
S P
SP
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors
D029721:Drosophila Proteins; C066696:ncd protein, Drosophila; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins