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.

A helical turn motif in Mss4 is a critical determinant of Rab binding and nucleotide release.

Monomeric Rab GTPases function as ubiquitous regulators of intracellular membrane trafficking. Mss4, an evolutionarily conserved Rab accessory factor, promotes nucleotide release from exocytic but not endocytic Rab GTPases. Here we describe the results of a high-resolution crystallographic and mutational analysis of Mss4. The 1.65 A crystal structure of Mss4 reveals a network of direct and water-mediated interactions that stabilize a partially exposed structural subdomain derived from four highly conserved but nonconsecutive sequence elements. The conserved subdomain contains the invariant cysteine residues required for Zn2+ binding as well as the residues implicated in the interaction with Rab GTPases. A strictly conserved DPhiPhi motif, consisting of an invariant aspartic acid residue (Asp 73) followed by two bulky hydrophobic residues (Met 74 and Phe 75), encodes a prominently exposed 3(10) helical turn in which the backbone is well-ordered but the side chains of the conserved residues are highly exposed and do not engage in intramolecular interactions. Substitution of any of these residues with alanine dramatically impairs nucleotide release activity toward Rab3A, indicating that the DPhiPhi motif is a critical element of the Rab interaction epitope. In particular, mutation of Phe 75 results in a defect as severe as that observed for mutation of Asp 96, which is located near the zinc binding site at the opposite end of the conserved subdomain. Despite severe defects, however, none of the mutant proteins is catalytically dead. Taken together, the results suggest a concerted mechanism in which distal elements of the conserved Rab interaction epitope cooperatively facilitate nucleotide release.

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.

Structural basis of activation and GTP hydrolysis in Rab proteins.

BACKGROUND: Rab proteins comprise a large family of GTPases that regulate vesicle trafficking. Despite conservation of critical residues involved in nucleotide binding and hydrolysis, Rab proteins exhibit low sequence identity with other GTPases, and the structural basis for Rab function remains poorly characterized. RESULTS: The 2. 0 A crystal structure of GppNHp-bound Rab3A reveals the structural determinants that stabilize the active conformation and regulate GTPase activity. The active conformation is stabilized by extensive hydrophobic contacts between the switch I and switch II regions. Serine residues in the phosphate-binding loop (P loop) and switch I region mediate unexpected interactions with the gamma phosphate of GTP that have not been observed in previous GTPase structures. Residues implicated in the interaction with effectors and regulatory factors map to a common face of the protein. The electrostatic potential at the surface of Rab3A indicates a non-uniform distribution of charged and nonpolar residues. CONCLUSIONS: The major structural determinants of the active conformation involve residues that are conserved throughout the Rab family, indicating a common mode of activation. Novel interactions with the gamma phosphate impose stereochemical constraints on the mechanism of GTP hydrolysis and provide a structural explanation for the large variation of GTPase activity within the Rab family. An asymmetric distribution of charged and nonpolar residues suggests a plausible orientation with respect to vesicle membranes, positioning predominantly hydrophobic surfaces for interaction with membrane-associated effectors and regulatory factors. Thus, the structure of Rab3A establishes a framework for understanding the molecular mechanisms underlying the function of Rab GTPases.

Gs alpha meets its target--shedding light on a key signal transduction event.

The recently determined crystal structure of Gs alpha bound to a catalytically active form of adenylyl cyclase reveals the location of the enzyme's active site and provides the first view of heterotrimeric G protein alpha subunit activating a downstream effector. Comparison with the structure of a catalytically inactive form of adenylyl cyclase suggests a plausible allosteric mechanism whereby the synergistic activators Gs alpha and forskolin stimulate the activity of adenylyl cyclase.

[Interhemispheric disconnection, Balint's syndrome and persistent anarthria: Marchifava-Bignami disease with white matter hemorrhage]. / Dysconnexion interhémisphérique, syndrome de Balint et troubles arthriques persistants: maladie de Marchiafava-Bignami avec hémorrhagie de la substance blanche.

A 37-year-old alcoholic right-handed man developed a complex neuropsychological picture following a mild head injury and a severe confusional state. Prominent features were Balint's syndrome, signs of interhemispheric deconnection, and speech disorders with anarthria and dysprosody. Iterative CT scans showed pathognomonic hypodensities of the genu and splenium of corpus callosum, confirmative of Machiafava-Bignami disease. After a two years follow-up, a favourable outcome was observed despite haemmoragic transformation of bilateral necrotic lesions of the parietal white matter, an exceptional neuropathological fact. This case is demonstrative of the possibility of articulate speech impairment when lesions of both corpus callosum and subcortical white matter are present. It also raises several aetiopathogenic problems which are discussed.

Chemical cross-linking of the substance P (NK-1) receptor to the alpha subunits of the G proteins Gq and G11.

We have previously shown that the high-affinity binding of substance P (SP) to its receptor is dependent on an interaction with a PTX-insensitive G protein. This G protein couples SP receptor activation to stimulation of its effector, phospholipase C. In this study, we combined photoaffinity labeling, chemical cross-linking techniques, and immunological characterization using sequence-specific antibody probes to identify G proteins that couple to the SP receptor. First we covalently labeled the SP receptor present on rat submaxillary gland membranes with a radioiodinated photoreactive derivative of SP, p-benzoyl-L-phenylalanine(8)-substance P (125I-[Bpa8]SP). Photoincorporation of this SP derivative was susceptible to guanine nucleotide inhibition, indicating that the receptor was coupled to its G protein during labeling. We then used a chemical cross-linking agent to covalently link the photoaffinity labeled SP receptor and its associated G protein. Cross-linking generated a 96 kDa product, formation of which was prevented by the addition of a guanine nucleotide, but not an adenine nucleotide, following photolabeling, but prior to cross-linking. Furthermore, the 96 kDa cross-linked complex was absent in membranes which had been depleted of G proteins by treatment with alkaline buffer prior to addition of the cross-linking agent. Reductive cleavage of the cross-link in the isolated 96 kDa complex yields two products: the 53 kDa SP receptor and a 42 kDa protein identified by immunoblot analysis as either G alpha q or G alpha 11. Antisera against a common sequence within G alpha s, G alpha i, and G alpha o showed no immunoreactivity to the complex or its cleavage products. These results provide the first direct evidence of specific interaction between photoaffinity labeled SP receptor and the alpha subunits of Gq and G11, members of a family of G proteins known to be associated with pertussis toxin-insensitive phospholipase C activation.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.