Structural basis for the interaction of the adaptor protein grb14 with activated ras.

Grb14, a member of the Grb7-10-14 family of cytoplasmic adaptor proteins, is a tissue-specific negative regulator of insulin signaling. Grb7-10-14 contain several signaling modules, including a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region, and a C-terminal Src-homology-2 (SH2) domain. We showed previously that the RA and PH domains, along with the BPS region and SH2 domain, are necessary for downregulation of insulin signaling. Here, we report the crystal structure at 2.4-Å resolution of the Grb14 RA and PH domains in complex with GTP-loaded H-Ras (G12V). The structure reveals that the Grb14 RA and PH domains form an integrated structural unit capable of binding simultaneously to small GTPases and phosphoinositide lipids. The overall mode of binding of the Grb14 RA domain to activated H-Ras is similar to that of the RA domains of RalGDS and Raf1 but with important distinctions. The integrated RA-PH structural unit in Grb7-10-14 is also found in a second adaptor family that includes Rap1-interacting adaptor molecule (RIAM) and lamellipodin, proteins involved in actin-cytoskeleton rearrangement. The structure of Grb14 RA-PH in complex with H-Ras represents the first detailed molecular characterization of tandem RA-PH domains bound to a small GTPase and provides insights into the molecular basis for specificity

Crystal Structure of the 65-Kilodalton Heat Shock Protein, Chaperonin 60.2, of Mycobacterium tuberculosis

Chaperonin 60s are a ubiquitous class of proteins that promote folding and assembly of other cellular polypeptides in an ATP-dependent manner. The oligomeric state of chaperonin 60s has been shown to be crucial to their role as molecular chaperones. Chaperonin 60s are also known to be important stimulators of the immune system. Mycobacterium tuberculosis possesses a duplicate set of chaperonin 60s, both of which have been shown to be potent cytokine stimulators. The M. tuberculosis chaperonin 60s are present in the extracellular milieu at concentrations that are extremely low for the formation of an oligomer. Here we present the crystal structure of one of the chaperonin 60s of M. tuberculosis, also called Hsp65 or chaperonin 60.2, at 3.2-Å resolution. We were able to crystallize the protein in its dimeric state. The unusual dimerization of the protein leads to exposure of certain hydrophobic patches on the surface of the protein, and we hypothesize that this might have relevance in binding to immunogenic peptides, as it does in the eukaryotic homologs

Crystal structure of Grb14^RA-PH-H-Ras and comparison to other RA-PH structures.

<p>(A) Ribbon diagram and molecular surface representation of the Grb14^RA-PH-H-Ras complex. The Grb14 RA domain is colored green, with β2 colored dark green, the PH domain is colored orange, with the phosphoinositide-binding β1-β2 loop colored magenta, and the RA-PH linker is colored gray. H-Ras is colored cyan, with switch 1 (residues 32-38) colored purple and switch 2 (residues 59-67) colored magenta. GTP is shown in stick representation with carbon atoms colored yellow, oxygen atoms red, nitrogen atoms blue, and phosphorus atoms black. The Mg²⁺ ion is colored brown. Select secondary-structure elements are labeled, along with the N- and C-termini. The view on the right is rotated by 90° as indicated. (B) Stereo diagram of a superposition of the structures (Cα traces) of Grb14^RA-PH, Grb10^RA-PH (PDB code 3HK0) [12], and RIAM^RA-PH (PDB code 3TCA) [17], in the same orientation as in the left panel of (A). Grb14^RA-PH is colored by domain as in (A), Grb10^RA-PH is colored blue (all) and RIAM^RA-PH is colored magenta (all). The N- and C-termini are labeled.</p

Comparison of Grb14^RA-PH-H-Ras structure with other RA domain-GTPase structures.

<p>(A) Stereo diagram of a superposition (aligned on Ras) of the structures (Cα traces) of Grb14^RA-PH-H-Ras and RalGDS^RA-H-Ras (PDB code 1LFD) [18]. Grb14^RA is colored green, RalGDS^RA is colored dark green, H-Ras is colored cyan (for Grb14^RA) or dark blue (for RalGDS^RA). Mg-GTP is shown bound to H-Ras in the Grb14^RA-PH structure. Select secondary-structure elements in the RA domains are labeled. (B) Stereo diagram of a superposition (aligned on Ras/Rap1) of the structures of Grb14^RA-PH-H-Ras and Raf1^RA-Rap1 (PDB code 1GUA) [26]. Raf1^RA is colored dark green and Rap1 is colored dark blue. Val88 of Raf1^RA is shown in stick and surface representation.</p

Cation-mediated interplay of loops in chaperonin-10

The ubiquitously occurring chaperonins consist of a large tetradecameric Chaperonin-60, forming a cylindrical assembly, and a smaller heptameric Chaperonin-10. For a functional protein folding cycle, Chaperonin-10 caps the cylindrical Chaperonin-60 from one end forming an asymmetric complex. The oligomeric assembly of Chaperonin-10 is known to be highly plastic in nature. In Mycobacterium tuberculosis, the plasticity has been shown to be modulated by reversible binding of divalent cations. Binding of cations confers rigidity to the metal binding loop, and also promotes stability of the oligomeric structure. We have probed the conformational effects of cation binding on the Chaperonin-10 structure through fluorescence studies and molecular dynamics simulations. Fluorescence studies show that cation binding induces reduced exposure and flexibility of the dome loop. The simulations corroborate these results and further indicate a complex landscape of correlated motions between different parts of the molecule. They also show a fascinating interplay between two distantly spaced loops, the metal binding "dome loop" and the GroEL-binding "mobile loop", suggesting an important cation-mediated role in the recognition of Chaperonin-60. In the presence of cations the mobile loop appears poised to dock onto the Chaperonin-60 structure. The divalent metal ions may thus act as key elements in the protein folding cycle, and trigger a conformational switch for molecular recognition

The 2.15 &#197; crystal structure of Mycobacterium tuberculosis chorismate mutase reveals an unexpected gene duplication and suggests a role in host-pathogen interactions

Chorismate mutase catalyzes the first committed step toward the biosynthesis of the aromatic amino acids, phenylalanine and tyrosine. While this biosynthetic pathway exists exclusively in the cell cytoplasm, the Mycobacterium tuberculosis enzyme has been shown to be secreted into the extracellular medium. The secretory nature of the enzyme and its existence in M. tuberculosis as a duplicated gene are suggestive of its role in host-pathogen interactions. We report here the crystal structure of homodimeric chorismate mutase (Rv1885c) from M. tuberculosis determined at 2.15 &#197; resolution. The structure suggests possible gene duplication within each subunit of the dimer (residues 35-119 and 130-199) and reveals an interesting proline-rich region on the protein surface (residues 119-130), which might act as a recognition site for protein-protein interactions. The structure also offers an explanation for its regulation by small ligands, such as tryptophan, a feature previously unknown in the prototypical Escherichia coli chorismate mutase. The tryptophan ligand is found to be sandwiched between the two monomers in a dimer contacting residues 66-68. The active site in the "gene-duplicated" monomer is occupied by a sulfate ion and is located in the first half of the polypeptide, unlike in the Saccharomyces cerevisiae (yeast) enzyme, where it is located in the later half. We hypothesize that the M. tuberculosis chorismate mutase might have a role to play in host-pathogen interactions, making it an important target for designing inhibitor molecules against the deadly pathogen

Crystallization and preliminary X-ray crystallographic studies of Mycobacterium tuberculosis chorismate mutase

Chorismate mutase from M. tuberculosis has been crystallized. Preliminary X-ray crystallographic studies reveal the occurrence of a dimeric molecule in the crystal asymmetric unit