Abstract
To study the relationship between conventional kinesin's structure and function, we identified 13 lethal mutations in the Drosophila kinesin heavy chain motor domain and tested a subset for effects on mechanochemistry. S246F is a moderate mutation that occurs in loop 11 between the ATP- and microtubule-binding sites. While ATP and microtubule binding appear normal, there is a 3-fold decrease in the rate of ATP turnover. This is consistent with the hypothesis that loop 11 provides a structural link that is important for the activation of ATP turnover by microtubule binding. T291M is a severe mutation that occurs in alpha-helix 5 near the center of the microtubule-binding surface. It impairs the microtubule-kinesin interaction and directly effects the ATP-binding pocket, allowing an increase in ATP turnover in the absence of microtubules. The T291M mutation may mimic the structure of a microtubule-bound, partially activated state. E164K is a moderate mutation that occurs at the beta-sheet 5a/loop 8b junction, remote from the ATP pocket. Surprisingly, it causes both tighter ATP-binding and a 2-fold decrease in ATP turnover. We propose that E164 forms an ionic bridge with alpha-helix 5 and speculate that it helps coordinate the alternating site catalysis of dimerized kinesin heavy chain motor domains.
