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Abstract

Rho GTPases are master spatial regulators of the cytoskeleton that control a wide range of cellular processes. Their inactivation by removal from cellular membranes involves the stepwise formation of a stable complex with guanine nucleotide dissociation inhibitors (RhoGDIs), for which process the RhoGDI N-terminus is indispensable. The formation of this interface has been thought to emerge from an intrinsically disordered state of RhoGDI in its free, apo form. Here we use state-of-the-art solution NMR methods, molecular dynamics simulations, and biochemical essays to pinpoint the site-specific structural features of full-length RhoGDI1 before and after binding its GTPase client Cdc42. In contrast to the current mechanistic understanding, a diverse set of NMR data unequivocally shows that the structural properties of the GDI N-terminus seen in crystal structures of the complex with GTPases already exist as largely preformed features in free, apo GDI. Even more interestingly, the required structural properties are imposed onto the terminus context-specifically by modulating interactions with the surface of the folded C-terminal domain. Lastly, upon Cdc42 binding, the flexibility of the N-terminus and its secondary-structural propensities are not largely abrogated. These observations change the textbook picture of the mechanism of membrane extraction of the GTPase. Rather than a disorder-to-order transition upon binding, an active role of the N-terminus with differentially preformed structural properties, suitably modulated by the specific surrounding along the multi-step process, seems required to leverage the intricate and highly selective extraction process.