Decoupling of nucleotide- and microtubule-binding sites in a kinesin mutant.
Molecular motors require ATP to move along microtubules or actin filaments. To understand how molecular motors function, it is crucial to know how binding of the motor to its filamentous track stimulates the hydrolysis of ATP by the motor, enabling it to move along the filament. A mechanism for the enhanced ATP hydrolysis has not been elucidated, but it is generally accepted that conformational changes in the motor proteins occur when they bind to microtubules or actin filaments, facilitating the release of ADP. Here we report that a mutation in the motor domain of the microtubule motor proteins Kar3 and Ncd uncouples nucleotide- and microtubule-binding by the proteins, preventing activation of the motor ATPase by microtubules. Unlike the wild-type motors, the mutants bind tightly to both ADP and microtubules, indicating that interactions between the nucleotide- and microtubule-binding sites are blocked. The region of the motor that includes the mutated amino acid could transmit or undergo a conformational change required to convert the motor ATPase into a microtubule-stimulated state.
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Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D003001:Cloning, Molecular; D029721:Drosophila Proteins; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D020409:Molecular Motor Proteins; D009154:Mutation; D020125:Mutation, Missense; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D029721:Drosophila Proteins; D005656:Fungal Proteins; C063504:KAR3 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C066696:ncd protein, Drosophila; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins