Kar3Vik1 mechanochemistry is inhibited by mutation or deletion of the C terminus of the Vik1 subunit.
Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.
From the Department of Biomedical and Molecular Sciences, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Monika
M
Joshi
Da
D
Duan
Doran
D
Drew
Zhimeng
Z
Jia
Darlene
D
Davis
Robert L
RL
Campbell
John S
JS
Allingham
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D041221:Candida glabrata / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits
ATPase; Kar3; Kinesin; Mechanotransduction; Microtubules; Molecular Dynamics; Molecular Motors; Motility; Neck Mimic; Vik1