TY - JOUR
T1 - The loop 5 element structurally and kinetically coordinates dimers of the human kinesin-5, Eg5
AU - Waitzman, Joshua S.
AU - Larson, Adam G.
AU - Cochran, Jared C.
AU - Naber, Nariman
AU - Cooke, Roger
AU - Jon Kull, F.
AU - Pate, Edward
AU - Rice, Sarah E.
N1 - Funding Information:
This work was supported by the National Institutes of Health under grant GM072656 (S.E.R., A.G.L., and N.N.), AR042895 (R.C. N.N.), GM077067 (E.P. and N.N.), and GM097079 (J.C.C. and F.J.K.). J.S.W. is supported by the Myhrvold Family Fellowship from the Fannie and John Hertz Foundation, the Malkin Scholars Program from the Robert H. Lurie Comprehensive Cancer Center of Northwestern University, and a Cellular and Molecular Basis of Disease training grant (GM08061).
PY - 2011/12/7
Y1 - 2011/12/7
N2 - 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.
AB - 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.
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U2 - 10.1016/j.bpj.2011.10.032
DO - 10.1016/j.bpj.2011.10.032
M3 - Article
C2 - 22261065
AN - SCOPUS:82955194449
SN - 0006-3495
VL - 101
SP - 2760
EP - 2769
JO - Biophysical Journal
JF - Biophysical Journal
IS - 11
ER -