Analysis of mutations in the kinesin motor that decouple ATPase activity and microtubule interaction
Auerbach, Scott David
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Conventional kinesin is a dimeric, microtubule-dependent motor whose activity is tightly coupled to ATP hydrolysis. Mutations that might affect the coupling between ATPase and motor activities of kinesin were predicted to fall within the gamma- phosphate sensor apparatus, a set of domains in the protein believed to detect the phosphorylation state of the bound nucleotide and mechanically transmit the information via conformational change to the microtubule-binding domain. An additional element, the relay helix, has been postulated to undergo axial translation, rotation, and/or elongation, in response to the loss of the gamma phosphate from the bound nucleotide, and serve as intermediary between nucleotide- and microtubule-binding sites. An N-terminal truncation of rat conventional kinesin was examined using steady- and transient-state kinetic methods. Rate constants for ATP and microtubule binding were determined, as well as those for microtubule-dependent ADP and phosphate release. The dimeric state of the motor in solution was confirmed using analytical ultracentrifugation. Conserved residues within the gamma phosphate sensor were selected for mutagenesis. The residue E237 is believed to form a transient salt bridge with R204 when the motor is in the ATP state, based on crystal structure analysis, and the mutations E237A and E237D were examined using transient state kinetic methods. Both mutants showed > 10-fold reduction in steady-state ATPase activity, although rate constants for ATP and microtubule binding, as well as ADP release were little affected. These results suggested a disruption in the catalysis step caused by the mutations. An electrostatic interaction between E200 and R204 may also form in response to changes in nucleotide phosphorylation state, however, E200D and E200A mutants were scarcely compromised in steady-state ATPase activity, and this was attributed to a reduction in the rate constants governing product release. Finally, the N256K mutation caused a >1000- fold reduction in the rate of ADP release and a ~100-fold reduction in the steady-state ATPase rate. N256 falls within the relay helix, although the mechanism by which the N256K defect arises cannot yet be determined.