A kinesin switch I arginine to lysine mutation rescues microtubule function.
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.
Klumpp
Lisa M
LM
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Mackey
Andrew T
AT
Farrell
Christopher M
CM
Rosenberg
John M
JM
Gilbert
Susan P
SP
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001665:Binding Sites; D002417:Cattle; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004331:Drosophila melanogaster; D005812:Genes, Switch; D006801:Humans; D006860:Hydrogen Bonding; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; D016547:Kinesins; D008239:Lysine