title: The loop 5 element structurally and kinetically coordinates dimers of the human kinesin-5, Eg5.
abstract: Eg5 is a homotetrameric kinesin-5 motor protein that generates outward force on the overlapping, antiparallel microtubules (MTs) of the mitotic spindle. Upon binding an MT, an Eg5 dimer releases one ADP molecule, undergoes a slow (∼0.5 s(-1)) isomerization, and finally releases a second ADP, adopting a tightly MT-bound, nucleotide-free (APO) conformation. This conformation precedes ATP binding and stepping. Here, we use mutagenesis, steady-state and pre-steady-state kinetics, motility assays, and electron paramagnetic resonance spectroscopy to examine Eg5 monomers and dimers as they bind MTs and initiate stepping. We demonstrate that a critical element of Eg5, loop 5 (L5), accelerates ADP release during the initial MT-binding event. Furthermore, our electron paramagnetic resonance data show that L5 mediates the slow isomerization by preventing Eg5 dimer heads from binding the MT until they release ADP. Finally, we find that Eg5 having a seven-residue deletion within L5 can still hydrolyze ATP and move along MTs, suggesting that L5 is not required to accelerate subsequent steps of the motor along the MT. Taken together, these properties of L5 explain the kinetic effects of L5-directed inhibition on Eg5 activity and may direct further interventions targeting Eg5 activity.
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois, USA.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Waitzman
authors.forename: Adam G
authors.initials: AG
authors.lastname: Larson
authors.forename: Jared C
authors.initials: JC
authors.lastname: Cochran
authors.forename: Nariman
authors.initials: N
authors.lastname: Naber
authors.forename: Roger
authors.initials: R
authors.lastname: Cooke
authors.forename: F
authors.initials: F
authors.lastname: Jon Kull
authors.forename: Edward
authors.initials: E
authors.lastname: Pate
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Rice
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D021381:Protein Transport; D012996:Solutions; D013329:Structure-Activity Relationship; D062367:ortho-Aminobenzoates / Q000378:metabolism
chemical_list: C470243:KIF11 protein, human; D015335:Molecular Probes; D050505:Mutant Proteins; D009711:Nucleotides; D012996:Solutions; D062367:ortho-Aminobenzoates; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; D000251:Adenosine Triphosphatases; D016547:Kinesins