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.

Lipoxygenases, which are widely distributed among plant and animal species, are Fe-containing dioxygenases that act on lipids containing (Z,Z)-pentadiene moieties in the synthesis of compounds with a variety of functions. Utilizing an improved strategy of data collection, low temperature, and synchrotron radiation of short wavelength, the structure of ferrous soybean lipoxygenase L-1, a single chain protein of 839 amino acid residues, has been determined by X-ray crystallography to a resolution of 1.4 A. The R-factor for the refined model is 19.7%. General features of the protein structure were found to be consistent with the results of prior crystallographic studies at lower (2.6 A) resolution. In contrast to the prior studies, the binding of a water molecule to the active site Fe was established. The octahedral coordination sphere of the Fe also includes the side chains of His499, His504, His690, and Asn694 as well as the terminal carboxylate of Ile839, which binds as a monodentate ligand. Asn694 is involved in a number of labile polar interactions with other protein groups, including an amide-aromatic hydrogen bond, and appears to be a weak ligand. Several possible access routes for dioxygen and fatty acids to the internal active site and substrate binding cavity are described. The protein structure restricts access to the Fe site such that the formation of an organo-Fe intermediate seems improbable. Structural restrictions pertinent to other proposed reaction intermediates, such as planar pentadienyl and nonplanar allyl radicals, are also discussed.

The Death Inducing Signaling Complex (DISC) formed by Fas receptor, FADD and caspase-8 is a pivotal trigger of apoptosis1-3. The Fas/FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains (DD) of Fas and FADD is at the center of DISC formation4, 5. Thus characterising the mechanistic basis for the Fas/FADD interaction is paramount for understanding DISC signaling but has remained enigmatic largely due to a lack of structural data. We have successfully formed and isolated the Fas/FADD DD complex and here we report the 2.7 Å crystal structure. The complex shows a tetrameric arrangement of four FADD DDs bound to four Fas DDs. We show that an opening of the Fas DD exposes the FADD binding site and simultaneously generates a Fas/Fas bridge. The result is a regulatory Fas/FADD complex bridge governed by weak protein:protein interactions revealing a model where the complex functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. Thus besides depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signaling solely by oligomerization and clustering events.

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The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

A high resolution crystal structure of Escherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 Å resolution. The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site. This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzymecatalyzed reaction. Escherichia coli alkaline phosphatase (AP)1 is a homodimeric metalloenzyme catalyzing the nonspecific hydrolysis of phosphate monoesters into inorganic phosphate and an alcohol. The overall structure is approximately 100 Å ϫ 50 Å ϫ 50 Å with the active sites 30 Å apart from each other on opposite sides of the molecule. Each active site contains two tightly bound zinc ions (Zn 1 and Zn 2 ) and one magnesium ion. The three closely spaced metal binding sites trace a triangle with the two zinc ions 4 Å apart and the magnesium binding site approximately 5 Å from Zn 2 and 7 Å from Zn 1 . The active site pocket shown in Fig. 1 (top panel) with bound inorganic phosphate is shallow and, in addition to the three metal ions, is lined by Arg-166 and Ser-102.Based on the structure of the enzyme with phosphate bound, Kim and Wyckoff (1) have proposed the reaction mechanism shown in Scheme 1. The free enzyme interacts with a phosphomonoester (ROP) to form the Michaelis enzyme-substrate complex (E⅐ROP). This complex breaks down upon nucleophilic attack of Ser-102 on the phosphate group of the substrate forming the covalent phospho-enzyme intermediate (E-P). This covalent intermediate is subsequently hydrolyzed into the noncovalent enzyme-phosphate complex (E⅐P i ). As predicted by the mechanism, the reaction proceeds with overall retention of configuration at the phosphorus center. The two consecutive in-line displacement steps are postulated to have trigonal bipyramidal transition states.In this study the interaction between vanadate (i.e. orthovanadate, VO 4 3Ϫ ) and E. coli alkaline phosphatase (EC 3.1.3.1) is evaluated by x-ray crystallography. Vanadate, an oxyanion of pentavalent vanadium, readily adopts a five-coordinate geometry resembling the proposed transition state in the enzyme-catalyzed reaction of alkaline phosphatase. In fact, vanadate has been used as a transition state analog in several enzyme-catalyzed reactions including phosphomonoester hydrolysis by rat acid phosphatase and ribonucleotide phosphodiester hydrolysis by ribonuclease A (2, 3). Vanadate is isostructural and isoelectronic with phosphate, a product, and a competitive inhibitor of alkaline phosphatase (4). Unlike the phosphate ion, vanadate can form stable five-coordinate species (4) allowing it to serve as a transition state analog for both the ac...

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...

Thionins belong to a rapidly growing family of biologically active peptides in the plant kingdom. Thionins are small ( approximately 5 kDA), cysteine-rich peptides with toxic and antimicrobial properties. They show a broad cellular toxicity against wide range of organisms and eukaryotic cell lines; while possessing some selectivity. Thionins are believed to be involved in protection against plant pathogens, including bacteria and fungi, by working directly at the membrane. The direct mechanism of action is still surrounded by controversy. Here the results of structural studies are reviewed and confronted with recent results of biophysical studies aimed at defining the function of thionins. The proposed toxicity mechanisms are reviewed and the attempt to reconcile competing hypotheses with a wealth of structural and functional studies is made.