title: Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling.
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.
authors.affiliation: Department of Biology, Jordan Hall, Indiana University, Bloomington, Indiana 47405, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Brendza
authors.forename: D J
authors.initials: DJ
authors.lastname: Rose
authors.forename: S P
authors.initials: SP
authors.lastname: Gilbert
authors.forename: W M
authors.initials: WM
authors.lastname: Saxton
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004330:Drosophila; D004789:Enzyme Activation; D020043:Genes, Essential; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009068:Movement; D020125:Mutation, Missense; D011485:Protein Binding
chemical_list: D020409:Molecular Motor Proteins; D000251:Adenosine Triphosphatases; D016547:Kinesins