Abstract
Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.
Highlights
The ATPase mechanism of kinesin requires that the active site hydrolyze ATP to ADP1⁄7Pi and communicate the nucleotide state at the active site to the microtubule to mediate specific conformational changes that generate movement
Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule
To understand the role of the switch I arginine in greater detail, we examined the kinetics of an arginine to lysine mutation at position 210, referred to as R210K
Summary
The neck linker has been shown to specify plus-end directionality of kinesin [25, 27,28,29,30] as well as docking to the catalytic core near the microtubule binding face, loop 12, and switch II relay helix ␣4 [25, 31] These results suggest a role for communication between the nucleotide and microtubule binding sites. Using the Drosophila conventional kinesin construct K401-wt, a mutant was constructed where the conserved arginine of switch I (Arg210 in the Drosophila melanogaster sequence) was changed to an alanine This mutant did not show a pre-steady-state burst of ADP1⁄7Pi product formation in acid quench experiments, indicative of a defect in ATP hydrolysis. The rate of ADP release from the second head of the Mt1⁄7R210A complex was similar using ATP, ADP, or AMP-PNP, whereas conventional wild-type kinesin shows discrimination among these different nucleotides in activating ADP release from the second head
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