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.

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...

These data--to our knowledge, for the first time--quantitatively show the presence, albeit at low levels, of two populations of heterosubtypic BnAbs against influenza A in human serum. These observations warrant further investigation to determine their origin, host polymorphism(s) that may affect their expression levels and how to boost these BnAb responses by vaccination to reach sustainable protective levels.

The association between the lipid bilayer and the membrane skeleton is important to cell function. In red blood cells, defects in this association can lead to various forms of hemolytic anemia. Although proteins involved in this association have been well characterized biochemically, the physical strength of this association is only beginning to be studied. Formation of a small cylindrical strand of membrane material (tether) from the membrane involves separation of the lipid bilayer from the membrane skeleton. By measuring the force required to form a tether, and knowing the contribution to the force due to the deformation of a lipid bilayer, it is possible to calculate the additional contribution to the work of tether formation due to the separation of membrane skeleton from the lipid bilayer. In the present study, we measured the tethering force during tether formation using a microcantilever (a thin, flexible glass fiber) as a force transducer. Numerical calculations of the red cell contour were performed to examine how the shape of the contour affects the calculation of tether radius, and subsequently separation work per unit area W(sk) and bending stiffness k(c). At high aspiration pressure and small external force, the red cell contour can be accurately modeled as a sphere, but at low aspiration pressure and large external force, the contour deviates from a sphere and may affect the calculation. Based on an energy balance and numerical calculations of the cell contour, values of the membrane bending stiffness k(c) = 2.0 x 10(-19) Nm and the separation work per unit area W(sk) = 0.06 mJ/m2 were obtained.

BackgroundThe NTF2-like superfamily is a versatile group of protein domains sharing a common fold. The sequences of these domains are very diverse and they share no common sequence motif. These domains serve a range of different functions within the proteins in which they are found, including both catalytic and non-catalytic versions. Clues to the function of protein domains belonging to such a diverse superfamily can be gleaned from analysis of the proteins and organisms in which they are found.ResultsHere we describe three protein domains of unknown function found mainly in bacteria: DUF3828, DUF3887 and DUF4878. Structures of representatives of each of these domains: BT_3511 from Bacteroides thetaiotaomicron (strain VPI-5482) [PDB:3KZT], Cj0202c from Campylobacter jejuni subsp. jejuni serotype O:2 (strain NCTC 11168) [PDB:3K7C], rumgna_01855) and RUMGNA_01855 from Ruminococcus gnavus (strain ATCC 29149) [PDB:4HYZ] have been solved by X-ray crystallography. All three domains are similar in structure and all belong to the NTF2-like superfamily. Although the function of these domains remains unknown at present, our analysis enables us to present a hypothesis concerning their role.ConclusionsOur analysis of these three protein domains suggests a potential non-catalytic ligand-binding role. This may regulate the activities of domains with which they are combined in the same polypeptide or via operonic linkages, such as signaling domains (e.g. serine/threonine protein kinase), peptidoglycan-processing hydrolases (e.g. NlpC/P60 peptidases) or nucleic acid binding domains (e.g. Zn-ribbons).

Cell cycle progression is controlled at several different junctures by the targeted destruction of cell cycle regulatory proteins. These carefully orchestrated events include the destruction of the securin protein to permit entry into anaphase, and the destruction of cyclin B to permit exit from mitosis. These destruction events are mediated by the ubiquitin/proteasome system. The human ubiquitin-conjugating enzyme, UbcH10, is an essential mediator of the mitotic destruction events. We report here the 1.95-Å crystal structure of a mutant UbcH10, in which the active site cysteine has been replaced with a serine. Functional analysis indicates that the mutant is active in accepting ubiquitin, although not as efficiently as wild-type. Examination of the crystal structure reveals that the NH 2 -terminal extension in UbcH10 is disordered and that a conserved 3 10 -helix places a lysine residue near the active site. Analysis of relevant mutants demonstrates that for ubiquitin-adduct formation the presence or absence of the NH 2 -terminal extension has little effect, whereas the lysine residue near the active site has significant effect. The structure provides additional insight into UbcH10 function including possible sites of interaction with the anaphase promoting complex/cyclosome and the disposition of a putative destruction box motif in the structure.Ubiquitin-mediated proteolysis regulates cell cycle progression at several key control points. At least two such control points occur in mitosis. One is at the transition from metaphase to anaphase and the other is at the exit from mitosis (for reviews, see Refs. 1-5). At the transition from metaphase to anaphase, the securin protein in the securin-separase protein complex is destroyed to release separase. The freed separase cleaves the protein complexes binding the sister chromatids together. Cleavage of these protein complexes is thought to facilitate sister chromatid segregation, and hence entry into anaphase. To exit from mitosis, cyclin B in the cyclin B-cdc2 complex must be destroyed. Destruction of cyclin B results in the inactivation of the cdc2 kinase. The inactivation of cdc2 is an essential event for resetting the cell cycle machinery (6).To accomplish the ubiquitination of securin and cyclin B (and other proteins targeted for ubiquitin-mediated destruction), three enzyme activities, designated E1 1 (ubiquitin activating enzyme), E2 (ubiquitin conjugating enzyme, Ubc), and E3 (ubiquitin ligase), must work in concert (for review, see Ref. 7). The E1 protein activates ubiquitin and then transfers it to the E2 protein. The ubiquitin forms an adduct to the E2 protein via a thiol ester linkage between the active site cysteine of E2 and the carboxyl terminus of ubiquitin. The E2 then donates the ubiquitin to the target protein, either directly or in conjunction with the E3 activity. In some instances, the same protein possesses both the E2 and E3 activity. Ultimately, a polyubiquitin-target protein conjugate is formed that then is recognized by the proteasome. The...

During maturation of the red blood cell (RBC) from the nucleated normoblast stage to the mature biconcave discocyte, both the structure and mechanical properties of the cell undergo radical changes. The development of the mechanical stability of the membrane reflects underlying changes in the organization of membraneassociated cytoskeletal proteins, and so provides an assessment of the time course of the development of membrane structural organization. Membrane stability in maturing erythrocytes was assessed by measuring forces required to form thin, tubular, lipid strands (tethers) from the surfaces of mononuclear cells obtained from fresh human marrow samples, marrow reticulocytes, circulating reticulocytes, and mature erythrocytes. Cells were biotinylated and manipulated with a micropipette to form an adhesive contact with a glass microcantilever, which gave a measure of the tethering force. The cell was withdrawn at controlled velocity and aspiration pressure to form a tether from the cell surface. The mean force required to form tethers from marrow reticulocytes and normoblasts was 27 ؎ 9 pN, compared to 54 ؎ 14 pN for mature cells. The energy of dissociation of the bilayer from the underlying skeleton increases 4-fold between the marrow reticulocyte stage and the mature cell, demonstrating that the mechanical stability of the membrane is not completely established until the very last stages of RBC maturation. IntroductionDuring the last stages of maturation of red blood cells (RBCs), dramatic changes occur in the structure and organization within the cell. The cell loses its nucleus, surface molecules are shed in small vesicles, and the final surface-to-volume ratio of the cell is established. [1][2][3] During this time, proteins that will eventually form the membrane-associated cytoskeleton (membrane skeleton) are synthesized and assembled at the intracellular surface of the plasma membrane. 4 The time course over which these protein assemblies become functionally viable is of interest, particularly with regard to hemolytic anemia and the early release of cells during hemorrhagic crisis, and could be important in designing methods for production of erythrocytes in vitro.The function of the assembled membrane skeleton is fundamentally mechanical, and therefore, studies of membrane mechanical properties in maturing cells provide the most direct assessment of the development of the functional viability of the skeleton during maturation. Early studies of membrane properties of both murine and human reticulocytes indicated increased membrane stiffness (shear rigidity) in those membranes. 5 This increased rigidity has been confirmed subsequently both by micropipette 6 and cell deformation in shear (ektacytometry). 7 In the latter study, evidence was also obtained that, despite increased mechanical stiffness, membranes of immature cells were less mechanically stable than their mature counterparts, as indicated by fragmentation of cells in fluid shear and in micropipette aspiration studies. The structural events...

The proteins securin and cyclin B are destroyed in mitosis by the ubiquitin/proteasome system. This destruction is important to mitotic progression. The N-terminal regions of these proteins contain the sequence features recognized by the ubiquitination system. We have demonstrated using circular dichroism and 1-D and 2-D nuclear magnetic resonance that these rather substantial regions are natively unfolded. Based on these ¢ndings, we propose a model that helps to explain previously enigmatic observations. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.