An allosteric propofol-binding site in kinesin disrupts kinesin-mediated processive movement on microtubules.

The Journal of Biological Chemistry
Kellie A WollRoderic G Eckenhoff

Abstract

Microtubule-based molecular motors mediate transport of intracellular cargo to subdomains in neurons. Previous evidence has suggested that the anesthetic propofol decreases the average run-length potential of the major anterograde transporters kinesin-1 and kinesin-2 without altering their velocity. This effect on kinesin has not been observed with other inhibitors, stimulating considerable interest in the underlying mechanism. Here, we used a photoactive derivative of propofol, meta-azipropofol (AziPm), to search for potential propofol-binding sites in kinesin. Single-molecule motility assays confirmed that AziPm and propofol similarly inhibit kinesin-1 and kinesin-2. We then applied AziPm in semiquantitative radiolabeling and MS microsequencing assays to identify propofol-binding sites within microtubule-kinesin complexes. The radiolabeling experiments suggested preferential AziPm binding to the ATP-bound microtubule-kinesin complex. The photolabeled residues were contained within the kinesin motor domain rather than at the motor domain-β-tubulin interface. No residues within the P-loop of kinesin were photolabeled, indicating an inhibitory mechanism that does not directly affect ATPase activity and has an effect on run lengt...Continue Reading

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Citations

Nov 22, 2019·The Journal of Biological Chemistry·Stephanie K DeebSusan P Gilbert
Jan 27, 2021·Proceedings of the National Academy of Sciences of the United States of America·Mandira DuttaBiman Jana
Nov 20, 2019·Current Biology : CB·Max B Kelz, George A Mashour

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Methods Mentioned

BETA
motility assay
motility studies
X-ray
affinity purification
gel filtration
protein assay

Software Mentioned

ZINC
NIH ImageJ
AxioVision
Computed Atlas of Surface Topography of proteins ( CASTp )
AutoDock Tools4
ImageJ
Mascot Daemon
PyMOL
GraphPad Prism
MarvinSketch

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