Multivalent endosome targeting by homodimeric EEA1.

Early endosome autoantigen localization to early endosomes is mediated by a C-terminal region, which includes a calmodulin binding motif, a Rab5 interaction site, and a FYVE domain that selectively binds phosphatidyl inositol 3-phosphate. The crystal structure of the C-terminal region bound to inositol 1,3-bisphosphate reveals an organized, quaternary assembly consisting of a parallel coiled coil and a dyad-symmetric FYVE domain homodimer. Structural and biochemical observations support a multivalent mechanism for endosomal localization in which domain organization, dimerization, and quaternary structure amplify the weak affinity and modest specificity of head group interactions with conserved residues. A unique mode of membrane engagement deduced from the quaternary structure of the C-terminal region provides insight into the structural basis of endosome tethering.

Determinants of the broad recognition of exocytic Rab GTPases by Mss4.

Rab GTPases function as essential regulators of vesicle transport between subcellular compartments of eukaryotic cells. Mss4, an evolutionarily conserved Rab accessory factor, facilitates nucleotide release and binds tightly to the nucleotide-free form of exocytic but not endocytic Rab GTPases. A structure-based mutational analysis of residues that are conserved only in exocytic Rab GTPases reveals three residues that are critical determinants of the broad specificity recognition of exocytic Rab GTPases by Mss4. One of these residues is located at the N-terminus of the switch I region near the nucleotide binding site whereas the other two flank an exposed hydrophobic triad previously implicated in effector recognition. The spatial disposition of these residues with respect to the structure of Rab3A correlates with the dimensions of the elongated Rab interaction epitope in Mss4 and supports a mode of interaction similar to that of other exchange factor-GTPase complexes. The complementarity of the corresponding interaction surfaces suggests a hypothetical structural model for the complex between Mss4 and Rab GTPases.

Structural plasticity of an invariant hydrophobic triad in the switch regions of Rab GTPases is a determinant of effector recognition.

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 beta sheet and the alpha2 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.