The structural basis for the divalent cation-dependent binding of heterodimeric alphabeta integrins to their ligands, which contain the prototypical Arg-Gly-Asp sequence, is unknown. Interaction with ligands triggers tertiary and quaternary structural rearrangements in integrins that are needed for cell signaling. Here we report the crystal structure of the extracellular segment of integrin alphaVbeta3 in complex with a cyclic peptide presenting the Arg-Gly-Asp sequence. The ligand binds at the major interface between the alphaV and beta3 subunits and makes extensive contacts with both. Both tertiary and quaternary changes are observed in the presence of ligand. The tertiary rearrangements take place in betaA, the ligand-binding domain of beta3; in the complex, betaA acquires two cations, one of which contacts the ligand Asp directly and the other stabilizes the ligand-binding surface. Ligand binding induces small changes in the orientation of alphaV relative to beta3.

Integrins are αβ heterodimeric receptors that mediate divalent cation-dependent cell-cell and cellmatrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin αVβ3 at 3.1 Å resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, αVβ3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed β-propeller from αV and an A domain from β3, and bears a striking resemblance to the Gα/Gβ interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the βA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and βA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.Integrins are large heterodimeric cell surface receptors found in many animal species ranging from sponges to mammals [reviewed in (1)]. These receptors are involved in fundamental cellular processes such as attachment, migration, proliferation, differentiation, and survival. Integrins also contribute to the initiation and/or progression of many common diseases including neoplasia, tumor metastasis, immune dysfunction, ischemia-reperfusion injury, viral infections, osteoporosis, and coagulopathies [reviewed in (2,3)]. An integrin is ~280 Å long and consists of one α (150 to 180 kD) and one β (~90 kD) subunit, both of which are type I membrane proteins. Eighteen α and eight β mammalian subunits are known, which assemble noncovalently into 24 different heterodimers. Contacts between the α and β subunits primarily involve their NH 2 -terminal halves [reviewed in (1)], which together form a globular head; the remaining portions form two rod-shaped tails (4-7) that span the plasma membrane.Like other receptors, integrins transmit signals to the cell interior (so-called "outside-in" signaling), which regulate organization of the cytoskeleton, activate kinase signaling cascades, and modulate the cell cycle and gene expression [reviewed in (8)]. Unlike other receptors, however, ligand binding with integrins is not generally constitutive but is regulated to reflect the activation state of the cell. This "inside-out" regulation of integrin affinity protects the host

Bacterial pathogens frequently use protein secretion to mediate interactions with their hosts. Here we found that a virulence locus (HSI-I) of Pseudomonas aeruginosa encodes a protein secretion apparatus. The apparatus assembled in discrete subcellular locations and exported Hcp1, a hexameric protein that forms rings with a 40 angstrom internal diameter. Regulatory patterns of HSI-I suggested that the apparatus functions during chronic infections. We detected Hcp1 in pulmonary secretions of cystic fibrosis (CF) patients and Hcp1-specific antibodies in their sera. Thus, HSI-I likely contributes to the pathogenesis of P. aeruginosa in CF patients. HSI-I-related loci are widely distributed among bacterial pathogens and may play a general role in mediating host interactions.Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the lungs of >80% of cystic fibrosis patients and is the primary cause of morbidity and mortality in these patients (1). A distinguishing feature of the bacterium is its high degree of versatility, which provides P. aeruginosa sufficient phenotypic plasticity to form both acute and chronic infections in humans (2,3). The choice between these disparate life-styles is governed by global virulence regulators, including RetS (regulator of exopolysaccharide and type III secretion) (4) and LadS (lost adherence sensor) (5). RetS and LadS reciprocally regulate virulence determinants such as type III secretion, which is RetS-activated and LadS-repressed, as well as exopolysaccharide production, which is RetS-repressed and LadS-activated. These virulence factors are important in acute and chronic infections, respectively.In addition to characterized virulence pathways, microarray analyses indicated that RetS and LadS reciprocally regulated a functionally uncharacterized virulence locus (Fig. 1). Consistent with its regulatory patterns by RetS and LadS, the virulence locus was required for chronic P. aeruginosa infection of the rat lung ( Fig. 1) homologous to a group of genes found in many Gram-negative proteobacteria that have been termed the IcmF-associated homologous protein (IAHP) cluster (7). P. aeruginosa encodes two other IAHP-related loci elsewhere in its genome; however, these loci were not regulated by either RetS or LadS and have no known role in virulence (Fig. 1). An overview of the distribution and genetic constituents of IAHP loci is shown (table S1).The IAHP-related locus of Vibrio cholerae, which the authors have designated a type VI secretion system, mediates cytotoxicity in phagocytic cells and is required for the extracellular secretion of four proteins lacking canonical hydrophobic amino-terminal signal sequences (8). We postulated that the RetS-and LadS-regulated IAHP locus in P. aeruginosa could play a similar role in extracellular protein targeting. To test this hypothesis, we activated expression of the locus in P. aeruginosa PAO1 by deleting retS. Comparison of the supernatant fractions of ΔretS and wild-type revealed that a small protein (M r ~1...

The crystal structure of Escherichia coli GroEL shows a porous cylinder of 14 subunits made of two nearly 7-fold rotationally symmetrical rings stacked back-to-back with dyad symmetry. The subunits consist of three domains: a large equatorial domain that forms the foundation of the assembly at its waist and holds the rings together; a large loosely structured apical domain that forms the ends of the cylinder; and a small slender intermediate domain that connects the two, creating side windows. The three-dimensional structure places most of the mutationally defined functional sites on the channel walls and its outward invaginations, and at the ends of the cylinder.

Insulin-degrading enzyme (IDE), a Zn2+-metalloprotease, is involved in the clearance of insulin and amyloid-beta (refs 1-3). Loss-of-function mutations of IDE in rodents cause glucose intolerance and cerebral accumulation of amyloid-beta, whereas enhanced IDE activity effectively reduces brain amyloid-beta (refs 4-7). Here we report structures of human IDE in complex with four substrates (insulin B chain, amyloid-beta peptide (1-40), amylin and glucagon). The amino- and carboxy-terminal domains of IDE (IDE-N and IDE-C, respectively) form an enclosed cage just large enough to encapsulate insulin. Extensive contacts between IDE-N and IDE-C keep the degradation chamber of IDE inaccessible to substrates. Repositioning of the IDE domains enables substrate access to the catalytic cavity. IDE uses size and charge distribution of the substrate-binding cavity selectively to entrap structurally diverse polypeptides. The enclosed substrate undergoes conformational changes to form beta-sheets with two discrete regions of IDE for its degradation. Consistent with this model, mutations disrupting the contacts between IDE-N and IDE-C increase IDE catalytic activity 40-fold. The molecular basis for substrate recognition and allosteric regulation of IDE could aid in designing IDE-based therapies to control cerebral amyloid-beta and blood sugar concentrations.

The crystal structure of the trp repressor/operator complex shows an extensive contact surface, including 24 direct and 6 solvent-mediated hydrogen bonds to the phosphate groups of the DNA. There are no direct hydrogen bonds or non-polar contacts to the bases that can explain the repressor's specificity for the operator sequence. Rather, the sequence seems to be recognized indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface. Water-mediated polar contacts to the bases also appear to contribute part of the specificity.

The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface. The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge. Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding. Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.

Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 A as a complex with the pheromone N-3-oxooctanoyl-L-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an alpha/beta/alpha sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix turn helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90 degree angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.