Mechanistic Analysis of Monomeric Kinesin Mutant K341-E164A

Thanh Ha
University of Pittsburgh

Conventional kinesin is a motor protein that binds and hydrolyzes ATP to generate plus-end directed movement along microtubules. Kinesin's pre-steady-state kinetics support a hand-over-hand mechanistic model in which the ATPase cycles of the two motor domains are slightly out-of-phase. This cooperativity between the motor domains would permit kinesin to traverse processively on microtubules. A Drosophila melanogaster genetic screen has identified glutamate 164 (E164) as a residue critical to kinesin function. When this residue is mutated to alanine (E164A), dimeric kinesin's ATPase activity is decreased by a factor of 4. This mutant binds ATP more tightly and hydrolyzes it more rapidly than wild-type. Kinetic data indicate that only one of the two motor domains can bind and hydrolyze ATP, and the mutant motor stalls on the microtubule. We have engineered a monomeric kinesin with the E164A substitution (K341-E164A) to determine the mechanistic basis of the stalling. The results indicate that the mutant dimer is affected primarily by defects that are intrinsic to the structural transitions in the motor domain, rather than simply by loss of cooperativity between the two motor domains of the dimer.