Plexins are single transmembrane receptors for semaphorin guidance cues, which regulate the cell motility through changes in the cells actin cytoskeleton. Since the actin cytoskeleton is regulated by small GTPases of the Rho family, interactions with small GTPases are of particular importance in the signaling mechanism of plexins. We previously mapped the interactions of small Rho GTPases, Rnd1 and active Rac1, with the plexin-B1 RBD to a common region of the structure, the Rho GTPase Binding Domain (RBD) [1]. NMR relaxation experiments on the RBD:Rac1 complex revealed that the Rac1 GTPases is less dynamic in the plexin bound state [2]. The current experiments show the effect of RBD : GTPase complex formation on amide hydrogen exchange from either protein. Amide hydrogen exchange �monitored either by solution NMR or mass spectrometry- is a technique that indicates the dynamics and stability of protein structures. Surprisingly, amides exchange more quickly from the Rnd1 GTPase when bound to the plexin-B1 RBD, suggesting that the GTPase is destabilized. This is in accord with previous thermodynamics measurements and suggests a difference in the mechanism of action of the two Rho GTPases, Rac1 and Rnd1, upon their interaction with plexin.[1] Hota, P.K., & Buck, M (2012) �Plexin structures are coming! Opportunities for multilevel investigations of the function of semaphoring guidance receptors, their cell signaling mechanisms and functions� Cell. & Mol. Life Sciences 69, 3765-805.[2] Bouguet-Bonnet, S. & Buck, M (2008) �Compensatory and long-range changes in ps-ns mainchain dynamics upon complex formation. 15N relaxation analysis of the free and bound states of the ubiquitin-like domain of human plexin-B1 and the small GTPase Rac1� J. Mol. Biol. 377, 1474-87.[3] Cao SF and Buck, M (2011) �Optimization and stabilization of Rho small GTPase proteins for solution NMR studies: The case of Rnd1� Small GTPases. 2(6), 295-304.

Binding of the Rho GTPase binding domain of plexin-B1 accelerates amide hydrogen exchange in the Rho GTPase Rnd1, indicating protein destabilization

248th National Meeting (2014)

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