Rab GTPases function as regulatory components of an evolutionarily conserved machinery that mediates docking, priming, and fusion of vesicles with intracellular membranes. We have previously shown that the active conformation of Rab3A is stabilized by a substantial hydrophobic interface between the putative conformational switch regions (Dumas, J. J., Zhu, Z., Connolly, J. L., and Lambright, D. G. (1999) Structure 7, 413-423). A triad of invariant hydrophobic residues at this switch interface (Phe-59, Trp-76, and Tyr-91) represents a major interaction determinant between the switch regions of Rab3A and the Rab3A-specific effector Rabphilin3A (Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374). Here, we report the crystal structure of the active form of Rab5C, a prototypical endocytic Rab GTPase. As is true for Rab3A, the active conformation of Rab5C is stabilized by a hydrophobic interface between the switch regions. However, the conformation of the invariant hydrophobic triad (residues Phe-58, Trp-75, and Tyr-90 in Rab5C) is dramatically altered such that the resulting surface is noncomplementary to the switch interaction epitope of Rabphilin3A. This structural rearrangement reflects a set of nonconservative substitutions in the hydrophobic core between the central  sheet and the ␣2 helix. These observations demonstrate that structural plasticity involving an invariant hydrophobic triad at the switch interface contributes to the mechanism by which effectors recognize distinct Rab subfamilies. Thus, the active conformation of the switch regions conveys information about the identity of a particular Rab GTPase as well as the state of the bound nucleotide.As general regulators of intracellular vesicle transport between donor and acceptor membranes, Rab proteins comprise the largest GTPase family with 11 distinct homologues in yeast and more than 50 known Rab GTPases in mammals (3-7). As is true of other GTPases of the Ras superfamily, Rabs cycle between active (GTP-bound) and inactive (GDP-bound) conformations (3,8,9). A key question concerns the molecular and structural mechanisms by which Rab GTPases generate specificity for a diverse spectrum of effectors and regulatory factors. Biochemical and genetic studies of chimeric and mutant Rab proteins have identified several hypervariable regions, including the N and C termini and the ␣3/5 loop, that play an important role in determining functional specificity (10, 11). However, interactions involving hypervariable regions cannot explain the ability of Rab GDI 1 (GDP dissociation inhibitor) to recognize most or all Rab GTPases yet still discriminate against other GTPase families or the ability of certain regulatory factors to recognize particular Rab subfamilies (12-15). These observations imply the existence of specificity determinants that are common to all Rab GTPases but not other GTPase families, as well as determinants that are conserved only within particular Rab subfamilies.Crystallographic studies of Rab GTPases have identified structural motifs a...
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