Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Thursday Speaker Abstracts

Tweaking the Kinesin-Microtubule Interface Daniel R. Peet 1 , Nigel Burroughs 2 , Robert A. Cross 1 . 1 Warwick Medical School, Coventry, United Kingdom, 2 Warwick University, Coventry, United Kingdom. The kinesin binding site lies entirely within a single tubulin heterodimer, and for some kinesins, unpolymerised tubulin heterodimers can fully activate the kinesin ATPase. Other kinesins require that tubulin assemble into microtubules before it can activate their ATPase - for example, the classical combination of brain tubulin and brain kinesin is like this. These behaviours show that kinesin sensitively reads the conformation of its binding site on tubulin. We have asked the obvious question, can motile kinesins feedback on the conformation of tubulin and on microtubule dynamics? We find that strong-state kinesin motor domains (apo or AMPPNP states of kinesin-1, or a rigor mutant, T93N) can dramatically alter microtubule dynamics. The action of kinesin-13 (MCAK) to destabilise the GTP-caps of dynamic microtubules is familiar; we find that strong-state binding of the kinesin-1 motor domain to microtubules has close to the opposite effect, stabilising the GDP-lattice against disassembly. GDP-microtubules are ordinarily extremely unstable and depolymerise endwise at ~200 heterodimers per second, via the unzipping and disassembly of curved GDP-protofilaments. The binding of strong-state kinesins reduces the off-rate of GDP-tubulins from the shrinking tip to <2 per second. The stabilising action of the kinesin and the disassembly rate of the microtubules can be controlled by titrating in GDP so that some of the kinesins are switched into weak binding. Further, if we anchor GDP- microtubules by their ends in a flow cell, and introduce strong-state kinesin motor domains via hydrodynamic flow, the microtubules bend in the flow, and kinesin binding locks their curvature. We speculate that kinesin does this by binding preferentially to the convex side of curved microtubules, stabilising thereby the increased lattice spacing and preventing recoil. With our cryoEM collaborators, Carolyn Moores and Ottilie von Loeffelholz, we are currently exploring whether kinesin binding can alter the subunit spacing of various microtubule lattices.

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