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

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 length without changing velocity. Our results also indicated that when the kinesin motor interacts with the microtubule during its processive run, a site forms in kinesin to which propofol can then bind and allosterically disrupt the kinesin–microtubule interaction, resulting in kinesin detachment and run termination. The discovery of the propofol-binding allosteric site in kinesin may improve our understanding of the strict coordination of the motor heads during the processive run. We hypothesize that propofol's potent effect on intracellular transport contributes to various components of its anesthetic action.

The ability of the kinesin-2 heterodimer KIF3AC to navigate microtubule networks is provided by the KIF3A motor domain

“…"Obtuse" intersections were defined as those in which the intersecting microtubules formed an angle greater than 90°. For these experiments, we included a similarly designed kinesin-1, KIF5B K439 as a control (54).…”

Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol atDrosophilaMotor Nerve Terminals

“…Recent work has shown that GABA-acting drugs such as propofol target a number of proteins other than GABA A receptors, including TrpA1 channels (Ton et al, 2017) and kinesin (Woll et al, 2018), resulting in parallel consequences on neural functions alongside sleep induction. Another emerging target mechanism is the presynaptic release machinery (van Swinderen and Kottler, 2014).…”

Tracking single molecule dynamics in the anesthetizedDrosophilabrain

“…Our results suggest a presynaptic mechanism for some of these drugs, which is consistent with other work done in cell culture and animal models (Bademosi et al, 2018b; Hemmings et al, 2005; Karunanithi et al, 2020; Zalucki et al, 2015). There is increasing evidence that general anesthetics may target mechanisms that influence presynaptic release efficacy, such as mitochondrial function (Morgan et al, 2002), kinesin assembly (Bensel et al, 2017; Woll et al, 2018), channel activation kinetics (Baumgart et al, 2015; Hemmings et al, 2005; Pavel et al, 2020; Zhou et al, 2019) and neurotransmission (Bademosi et al, 2018b; Karunanithi et al, 2020; Troup et al, 2019; Zalucki et al, 2015). More broadly, tracking single particle dynamics in ex vivo brains of adult Drosophila flies opens a new window into understanding the behavior of individual molecules in intact tissue, to for example help determine which mechanisms are drug-specific and which might reflect a common property of diverse drugs.…”