The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner

• Published in: The Journal of biological chemistry Vol. 292; no. 35; pp. 14680 - 14694
• Main Authors: Bell, Kayla M., Cha, Hyo Keun, Sindelar, Charles V., Cochran, Jared C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2017; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Diphosphate - chemistry; Adenosine Diphosphate - metabolism; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Adenylyl Imidodiphosphate - chemistry; Adenylyl Imidodiphosphate - metabolism; Amino Acid Substitution; ATPase; Binding Sites; Binding, Competitive; Biocatalysis; cryo-electron microscopy; Cryoelectron Microscopy; Crystallography, X-Ray; enzyme mechanism; Gene Deletion; Humans; kinesin; Kinesin - chemistry; Kinesin - genetics; Kinesin - metabolism; kinetics; microtubule; Microtubules - chemistry; Microtubules - enzymology; Microtubules - metabolism; Models, Molecular; Molecular Biophysics; molecular motor; Mutation; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Conformation; Protein Interaction Domains and Motifs; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; thermodynamics; Tubulin - chemistry; Tubulin - metabolism; microtubule; ATPase; kinesin; thermodynamics; enzyme mechanism; kinetics; Saccharomyces cerevisiae; molecular motor; cryo-electron microscopy
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.797662

Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αβ-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single “canonical” site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate (“noncanonical”) sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αβ-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal.