Influenza virus remains a constant public health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Monoclonal antibody (mAb)-based immunotherapy is a promising strategy for disease control. Here we use a human Ab phage display library and H5 hemagglutinin (HA) ectodomain to select ten neutralizing mAbs (nAbs) with a remarkably broad range among Group 1 influenza viruses, including the H5N1 “bird flu” and the H1N1 “Spanish flu” strains. Notably, nine of the Abs utilize the same germline gene, VH1-69. The crystal structure of one mAb bound to H5N1 HA reveals that only the heavy chain inserts into a highly conserved pocket in the HA stem, inhibiting the conformational changes required for membrane fusion. Our studies indicate that nAbs targeting this pocket could provide broad protection against both seasonal and pandemic influenza A infections.

Control of integrin affinity for ligands (integrin activation) is essential for normal cell adhesion, migration, and assembly of an extracellular matrix. Integrin activation is usually mediated through the integrin beta subunit cytoplasmic tail and can be regulated by many different biochemical signaling pathways. We report that specific binding of the cytoskeletal protein talin to integrin beta subunit cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that increase their affinity. Thus, regulated binding of talin to integrin beta tails is a final common element of cellular signaling cascades that control integrin activation.

We have determined the high resolution crystal structure of the A domain from the alpha chain of integrin CR3. The domain adopts a classic alpha/beta "Rossmann" fold and contains an unusual Mg2+ coordination site at its surface. One of the coordinating ligands is the glutamate side chain from another A domain molecule. We suggest that this site represents a general metal ion-dependent adhesion site (MIDAS) for binding protein ligands. We further propose that the beta subunits of integrins contain a MIDAS motif within a modified A domain. Our crystal structure will allow reliable models to be built for other members of the A domain superfamily and should facilitate development of novel adhesion modulatory drugs.

We have determined the crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition.

Protective antigen (PA) is the central component of the three-part protein toxin secreted by Bacillus anthracis, the organism responsible for anthrax. After proteolytic activation on the host cell surface, PA forms a membrane-inserting heptamer that translocates the toxic enzymes, oedema factor and lethal factor, into the cytosol. PA, which has a relative molecular mass of 83,000 (M(r) 83K), can also translocate heterologous proteins, and is being evaluated for use as a general protein delivery system. Here we report the crystal structure of monomeric PA at 2.1 A resolution and the water-soluble heptamer at 4.5 A resolution. The monomer is organized mainly into antiparallel beta-sheets and has four domains: an amino-terminal domain (domain 1) containing two calcium ions and the cleavage site for activating proteases; a heptamerization domain (domain 2) containing a large flexible loop implicated in membrane insertion; a small domain of unknown function (domain 3); and a carboxy-terminal receptor-binding domain (domain 4). Removal of a 20K amino-terminal fragment from domain 1 allows the assembly of the heptamer, a ring-shaped structure with a negatively charged lumen, and exposes a large hydrophobic surface for binding the toxic enzymes. We propose a model of pH-dependent membrane insertion involving the formation of a porin-like, membrane-spanning beta-barrel.

Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins.

Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of neurodegenerative diseases and stroke. However, the mechanism of MMP activation remains unclear. We report that MMP activation involves S-nitrosylation. During cerebral ischemia in vivo, MMP-9 colocalized with neuronal nitric oxide synthase. S-Nitrosylation activated MMP-9 in vitro and induced neuronal apoptosis. Mass spectrometry identified the active derivative of MMP-9, both in vitro and in vivo, as a stable sulfinic or sulfonic acid, whose formation was triggered by S-nitrosylation. These findings suggest a potential extracellular proteolysis pathway to neuronal cell death in which S-nitrosylation activates MMPs, and further oxidation results in a stable posttranslational modification with pathological activity.

The crystallographically determined structure of simian virus 40 shows that the 72 pentamers of viral protein VP1, which form the outer shell, have identical conformations except for the C-terminal arms of their subunits. Five arms emerge from each pentamer and insert into neighbouring pentamers. This tying together of standard building blocks allows for the required variability in packing geometry without sacrificing specificity.