Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling

To study the relationship between conventional kinesin's structure and func tion, we identified 13 lethal mutations in the Drosophila kinesin heavy cha in motor domain and tested a subset for effects on mechanochemistry, S246F is a moderate mutation that occurs in loop 11 between the ATP- and microtub ule-binding sites. While ATP and microtubule binding appear normal, there i s a 3-fold decrease in the rate of ATP turnover, This is consistent with th e hypothesis that loop 11 provides a structural link that is important for the activation of ATP turnover by microtubule binding. T291M is a severe mu tation that occurs in alpha-helix 5 near the center of the microtubule-bind ing surface. It impairs the microtubule-kinesin interaction and directly ef fects the ATP-binding pocket, allowing an increase in ATP turnover in the a bsence of microtubules. The T291M mutation may mimic the structure of a mic rotubule-bound, partially activated state. E164K is a moderate mutation tha t 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 di merized kinesin heavy chain motor domains.