Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd.
Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor domains is to analyze the ATPase cycle of dimeric MC1 and monomeric MC6. The steps in the ATPase cycle where cooperativity occurs can be identified by comparing the two mechanisms. The rate-limiting step in the MC6 mechanism is ADP release at 3.4 s(-)(1). The observed rate constant for ATP-induced dissociation from the microtubule is 14 s(-)(1). However, the relative amplitude associated with MC6 dissociation is extremely small in comparison to the amplitude associated with dimeric MC1 dissociation kinetics. The amplitude data indicate that monomeric MC6 does not detach from the microtubule during the initial turnovers of ATP, and ATP hydrolysis is uncoupled from movement. The results show that cooperative interactions between the motor domains of the dimer are required for ATP-dependent dissociation; therefore, one function of the partner motor domain may be to weaken the interaction of the adjacent head with the microtubule.
Mackey
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Department of Biological Sciences, 518 Langley Hall, University of Pittsburgh, PA 15260, USA.
Gilbert
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SP
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019281:Dimerization; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005456:Fluorescent Dyes / Q000378:metabolism; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D062367:ortho-Aminobenzoates / Q000378:metabolism
D000345:Affinity Labels; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D020409:Molecular Motor Proteins; C066696:ncd protein, Drosophila; D062367:ortho-Aminobenzoates; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins