The conserved L5 loop establishes the pre-powerstroke conformation of the Kinesin-5 motor, eg5.
Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.
Department of Cell and Molecular Biology, Northwestern University, Chicago, Illinois, USA.
Adam G
AG
Larson
Nariman
N
Naber
Roger
R
Cooke
Edward
E
Pate
Sarah E
SE
Rice
D000494:Allosteric Regulation; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008870:Microtubules / Q000737:chemistry; D008958:Models, Molecular; D009038:Motion; D011487:Protein Conformation
C003724:3-tritylthio-L-alanine; D016547:Kinesins; D003545:Cysteine