Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
Alex F Thompson
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, United States
April Alfieri
Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, United States
Ignas Gaska
Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, United States
Jennifer Major
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Lorain, United States; Department of Pharmacology, Mayo Clinic, Jacksonville, United States
Garrett Debs
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
Sayaka Inagaki
Department of Pharmacology, Mayo Clinic, Jacksonville, United States
Pedro Gutierrez
Department of Molecular and Cellular Biology, University of California, Davis, Davis, United States
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, United States
Ronald Milligan
Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, United States
Jason Stumpff
Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, United States
Steven S Rosenfeld
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Lorain, United States; Department of Pharmacology, Mayo Clinic, Jacksonville, United States
Scott T Forth
Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, United States
Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.