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.

Anthrax toxin consists of the proteins protective antigen (PA), lethal factor (LF) and oedema factor (EF). The first step of toxin entry into host cells is the recognition by PA of a receptor on the surface of the target cell. Subsequent cleavage of receptor-bound PA enables EF and LF to bind and form a heptameric PA63 pre-pore, which triggers endocytosis. Upon acidification of the endosome, PA63 forms a pore that inserts into the membrane and translocates EF and LF into the cytosol. Two closely related host cell receptors, TEM8 and CMG2, have been identified. Both bind to PA with high affinity and are capable of mediating toxicity. Here, we report the crystal structure of the PA-CMG2 complex at 2.5 A resolution. The structure reveals an extensive receptor-pathogen interaction surface mimicking the non-pathogenic recognition of the extracellular matrix by integrins. The binding surface is closely conserved in the two receptors and across species, but is quite different in the integrin domains, explaining the specificity of the interaction. CMG2 engages two domains of PA, and modelling of the receptor-bound PA63 heptamer suggests that the receptor acts as a pH-sensitive brace to ensure accurate and timely membrane insertion. The structure provides new leads for the discovery of anthrax anti-toxins, and should aid the design of cancer therapeutics.

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV).The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 Å resolution, as well as the structure of the uncomplexed S1 RBD at 2.2 Å resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor-and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.Severe acute respiratory syndrome (SARS), 3 a newly emerged infectious disease, claimed 813 lives from ϳ8000 patients during a 2003 global epidemic. In severe illness, influenza-like symptoms quickly progress to pneumonia, hypoxia, and acute respiratory distress and failure, resulting in 10% overall death rate with exceptionally high mortality among the elderly (1). A novel coronavirus (SARS-CoV) has been identified as the etiological agent of SARS. The SARS-CoV surface spike protein S mediates viral entry into the host cell (2) and includes two functional domains as follows: S1 (Gly 13 -Arg 667 ) and S2 (Ser 668 -Thr 1255 ). S1 contains the host-specific receptor binding domain (RBD), whereas S2 mediates fusion between viral and host cell membranes (3). Angiotensin-converting enzyme 2 (ACE2) was identified as a functional receptor for the SARSCoV (4). The recently determined structure of the S1-RBD in complex with the extracellular domain of ACE2 (5) illustrates the structural basis for the initial step of virus-host recognition.As the mediator of host-specific SARS infection and a major viral surface antigen, the S protein is an attractive candidate for both vaccine development and immunotherapy. Marasco and co-workers (6) previously identified a potent neutralizing human monoclonal antibody against the S1 RBD, designated "80R," from two nonimmune (i.e. not restricted by B cell recombination) human antibody libraries. 80R binds S1 with nanomolar affinity, blocks the binding of S1 to ACE2, prevents the formation of syncytia in vitro (6), and inhibits viral replication in vivo (7). Deletion studies have shown that the 80R epitope on S1 is located in the minimal ACE2 binding domain, between residues 324 and 503 (6, 7).Here, we rep...

No abstract

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type I␥ (PIPKI␥), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKI␥ C terminus in a mode very similar to that of integrin binding. However, PIPKI␥ binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKI␥. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the fulllength PIPKI␥. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKI␥ binding that regulates focal adhesion turnover.

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets ␤-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating ␤-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.Polyubiquitination of specific proteins in cells involves the concerted action of E1, 5 E2, and E3 enzymes. First, E1 covalently binds and activates ubiquitin for subsequent transfer to one of several E2s. The latter can in turn directly transfer its bound ubiquitin to the amino groups of lysine side chains in target proteins. More often, however, E3 ligases recognize substrates and direct their interaction with E2s, resulting in the highly specific regulation of target protein polyubiquitination (1, 2). Humans carry two highly related genes, siah1 and siah2 (3), that encode the mammalian homologs of the Drosophila Sina protein, which is required for R7 photoreceptor cell differentiation within the sevenless pathway (4, 5). Sina/Siah proteins are E3 ligases, acting either as single proteins or as part of a multiprotein complex that is analogous to the Skp1-cullin-1-F-box (SCF) complex. Among the targets of Sina/Siah are NcoR (6), DCC (7), c-Myb (8), BOB-1/OBF-1 (9, 10), Peg3/Pw1 (11), Kid (12), Numb (13) (24), and ␣-ketoglutarate dehydrogenase (25). In addition, Siah interacts with adenomatous polyposis coli, a tumor suppressor involved in colon cancers (26); VAV, a nucleotide exchange factor involved in control of Rho/Rac proteins (27); BAG-1, a Hsp70/Hsc70-binding protein that modulates pathways involved in the control of cell proliferation, death, and migration (28, 29); and Dab-1, an inhibitor of Siah1 (30). However, Sina/Siah does not appear to target phyllopod, adenomatous polyposis coli, VAV, BAG-1, or Dab-1 for polyubiquitination and degradation. Thus, not all Siahbinding proteins are targets of Siah-mediated degradation.Recently, we discovered a novel pathway for ␤-catenin degradation involving a complex formed by Siah1, SIP, the adaptor protein Skp1 that is common to the SCF complex, and the F-box protein Ebi that binds ␤-catenin independent of phosphorylation (31). Siah1 expression is upregulated by p53, revealing a link between genotoxic injury and destruction of ␤-catenin, reduced Tcf/LEF activity, and cell cycle arrest (31). Siah1 is a dimeric protein that contains an N-terminal RING domain (an E2 binding domain) followed by...

No abstract

The E3 ubiquitin ligase Siah regulates key cellular events that are central to cancer development and progression. A promising route to Siah inhibition is disrupting its interactions with adaptor proteins. However, typical of protein-protein interactions, traditional unbiased approaches to ligand discovery did not produce viable hits against this target, despite considerable effort and a multitude of approaches. Ultimately, a rational structure-based design strategy was successful for the identification of novel Siah inhibitors in which peptide binding drives specific covalent bond formation with the target. X-ray crystallography, mass spectrometry and functional data demonstrate that these peptide-mimetics are efficient covalent inhibitors of Siah and antagonize Siah-dependent regulation of Erk and Hif signaling in cell. The strategy proposed may result useful as a general approach to the design of peptide-based inhibitors of other protein-protein interactions.