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bioRxiv · 10.1101/2022.07.18.500463

GTPase-dependent cyclic flexibility transitions drive the two-component EEA1-Rab5 molecular motor

Singh, A.·Soler, J. A.·Lauer, J.·Grill, S. W.·Jahnel, M.·Zerial, M.·Thutupalli, S.
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Abstract

The recognition of vesicles by their correct target compartment depends on the pairing of small GTPases with effector proteins (1-3). Whereas ATPases can cyclically convert the free energy of ATP hydrolysis into mechanical work, the function of small GTPases is predominantly associated with signal transduction processes, and their role in mechano-transduction is less established (4-6). However, binding of the GTPase Rab5 to the long coiled-coil tethering protein EEA1 on an early endosome induces a rigidity transition resulting in a large conformational change in EEA1 from a rigid and extended to a flexible and collapsed state. This entropic collapse of EEA1 gives rise to an effective force that can pull tethered membranes closer (7). It currently remains unclear if EEA1 can return from the collapsed to the extended conformation without the aid of chaperones. Here, we use fluorescence correlation spectroscopy to reveal that EEA1 in bulk solution can undergo multiple flexibility transition cycles that are associated with the binding and release of Rab5(GTP) and Rab5(GDP). Using semi-flexible polymer theory we provide evidence that the cyclic transitioning of Rab5-EEA1 between extended and collapsed conformations is driven by the energetics of Rab5 binding/unbinding and GTP hydrolysis. Cyclic flexibility transitions represent a complete mechanical work cycle that is able to perform up to 20 kBT of mechanical work per cycle, against an opposing force. Hence, Rab5 and EEA1 constitute a two-component molecular motor driven by the chemical energy derived from GTP hydrolysis by Rab5. We conclude that coiled-coil tethering proteins and their small GTPase partners can have active mechanical roles in membrane trafficking.

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Singh, A., Soler, J. A., Lauer, J., Grill, S. W., Jahnel, M., Zerial, M., Thutupalli, S.. 2022-07-19. GTPase-dependent cyclic flexibility transitions drive the two-component EEA1-Rab5 molecular motor. https://doi.org/10.1101/2022.07.18.500463

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