title: Loop L5 assumes three distinct orientations during the ATPase cycle of the mitotic kinesin Eg5: a transient and time-resolved fluorescence study.
abstract: Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the "static" images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis.
authors.affiliation: From the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Muretta
authors.forename: William M
authors.initials: WM
authors.lastname: Behnke-Parks
authors.forename: Jennifer
authors.initials: J
authors.lastname: Major
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Petersen
authors.forename: Adeline
authors.initials: A
authors.lastname: Goulet
authors.forename: Carolyn A
authors.initials: CA
authors.lastname: Moores
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.forename: Steven S
authors.initials: SS
authors.lastname: Rosenfeld
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D001665:Binding Sites; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000737:chemistry / Q000648:ultrastructure*; D008938:Mitosis / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: C470243:KIF11 protein, human; D000266:Adenylyl Imidodiphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins
keywords: Anisotropy Decay; Cryo-EM; Eg5; Electron Microscopy (EM); Fluorescence; Kinesin; Kinetics; Microtubules; Stopped Flow; Time-resolved Fluorescence