Intramolecular signaling upon complexation

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Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.antibody | cryoelectron microscopy | flavivirus W est Nile virus (WNV) is a human pathogen that causes a febrile illness, which can progress to encephalitis, paralysis, and death. The virus is endemic in parts of Africa, Asia, and Europe, and in the past decade has spread throughout North America and into Central and South America (1). WNV is closely related to other arthropod-transmitted, medically relevant flaviviruses, such as dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses. These lipid-enveloped viruses enter host cells by receptor-mediated endocytosis. The singlestranded, positive-sense RNA genome is released into the cytoplasm after low pH induces the fusion of the viral lipid envelope with the endosomal membrane.Mature WNV virions are roughly spherical with a diameter of about 500 Å. The outer viral surface is composed of an icosahedral scaffold of 180 closely packed copies of the membrane-anchored envelope (E) glycoprotein. Sets of three, nearly parallel E homodimers are associated into rafts that form a herringbone pattern on the surface of mature virions. The ectodomain of E has three structural domains, DI, DII, and DIII (2-5), with domain DI positioned structurally between DII and DIII. DII contains a fusion loop at its distal end that is indispensable for virus-cell membrane fusion. The Ig-like C-terminal domain DIII undergoes a major, pH-triggered positional rearrangement essential for fusion, and may also be involved in receptor binding (2, 6-12). During cell entry of flaviviruses, low endosomal pH triggers a proposed E protein rearrangement cascade, including the dissociation of E dimers and the outward rotation of DII during the repositioning of E monomers into fusion-active trimers (6, 9, 13).The humoral immune response is essential for protection against flavivirus infection and disease (14,15). The E glycoprotein is the principal antigen that elicits neutralizing antibodies agai...

Section: Resultsmentioning

confidence: 55%

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

Kaufmann

Vogt²,

Goudsmit³

et al. 2010

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121

“…Indeed, a conformational change in the elbow angle of Ϸ40°relative to that in the crystal structure of the Fab E16ϩDIII complex (Fig. 4) shows flexibility, as is often found in antibody structures (30)(31)(32)(33). Presumably, the very similar elbow angles in the Fab molecules bound to the independent binding sites DIII-B and DIII-C in the cryo-EM structure represent the lowest energy conformation, whereas the x-ray structure may have a slightly higher energy to achieve better crystal packing.…”

Section: W Est Nile Virus (Wnv) Causes a Febrile Illness In Humansmentioning

confidence: 64%

West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

Kaufmann

Nybakken

Chipman

et al. 2006

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122

Flaviviruses, such as West Nile virus (WNV), are significant human pathogens. The humoral immune response plays an important role in the control of flavivirus infection and disease. The structure of WNV complexed with the Fab fragment of the strongly neutralizing mAb E16 was determined to 14.5-Å resolution with cryoelectron microscopy. E16, an antibody with therapeutic potential, binds to domain III of the WNV envelope glycoprotein. Because of steric hindrance, Fab E16 binds to only 120 of the 180 possible binding sites on the viral surface. Fitting of the previously determined x-ray structure of the Fab-domain III complex into the cryo-electron microscopy density required a change of the elbow angle between the variable and constant domains of the Fab. The structure suggests that the E16 antibody neutralizes WNV by blocking the initial rearrangement of the E glycoprotein before fusion with a cellular membrane.cryo-electron microscopy ͉ flavivirus ͉ neutralization W est Nile virus (WNV) causes a febrile illness in humans that can progress to encephalitis, paralysis, and death. Although endemic in parts of Africa, Asia, Europe, and the Middle East, it was first isolated in the United States in 1999 and has subsequently spread throughout North America and the Caribbean (1-3). The similar Kunjin virus is found in Australia (4). WNV, a member of the Flaviviridae family, is closely related to other arthropod-borne, medically important viruses, such as dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses. These small, Ϸ500-Å-diameter, lipidenveloped viruses enter their host cells by receptor-mediated endocytosis. A low pH-triggered conformational rearrangement of the viral surface glycoproteins resulting in a fusion-active state of the virion allows the release of the single-stranded, positivesense RNA genome from the endosomes into the cytoplasm.The outer surface of mature infectious particles is formed by an icosahedral scaffold of 90 homodimers of the membraneanchored envelope glycoprotein (E). The three E monomers per icosahedral asymmetric unit each have distinctly different environments (5, 6), contrary to most other icosahedral viruses in which all subunits have at least quasi-similar environments. The ectodomain of E consists of three structural domains, DI, DII, and DIII (7-9). DI is structurally positioned between DII and DIII. The dimerization DII contains a 12-residue-long loop essential for virus-cell membrane fusion and, in dengue virus, has carbohydrate moieties that mediate receptor binding (10). The C-terminal DIII undergoes a major, pH-triggered, positional rearrangement essential for fusion and may also be involved in receptor binding (7,(11)(12)(13)(14)(15)(16)(17)(18).The humoral immune response is crucial for protection against flavivirus infection and disease (19). Antibodies can prevent infection by interference with functions mediated by viral surface proteins, such as receptor attachment, virus internalization, and membrane fusion. Indeed, the E glycoprotein is the...

“…Amino acid substitution experiments have shown that disrupting the ball-and-socket joint can markedly decrease signaling while having only a minimal effect on antigen binding (24). Antigen binding is thought to cause the elbow to bend, affecting the initiation of signaling (25,26). However, precisely how bending relates to signaling remains unclear; indeed, the mechanism of signaling itself remains uncertain, with competing proposals disagreeing over whether aggregation or disaggregation of BCRs at the B-cell surface is required (27)(28)(29).…”

Section: Discussionmentioning

confidence: 99%

Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells

Kaplinsky

Sun

et al. 2014

Antibody repertoires are known to be shaped by selection for antigen binding. Unexpectedly, we now show that selection also acts on a non-antigen-binding antibody region: the heavy-chain variable (V H )-encoded "elbow" between variable and constant domains. By sequencing 2.8 million recombined heavy-chain genes from immature and mature B-cell subsets in mice, we demonstrate a striking gradient in V H gene use as pre-B cells mature into follicular and then into marginal zone B cells. Cells whose antibodies use V H genes that encode a more flexible elbow are more likely to mature. This effect is distinct from, and exceeds in magnitude, previously described maturation-associated changes in heavy-chain complementarity determining region 3, a key antigen-binding region, which arise from junctional diversity rather than differential V H gene use. Thus, deep sequencing reveals a previously unidentified mode of B-cell selection.immunomics | principal component analysis | development T he mature antibody repertoire is shaped by selective forces that influence B-cell survival (1). Comparison of immature and mature B-cell subsets has shown that selection acts specifically on complementarity determining region 3 (CDR3) of the antibody heavy-chain molecule, an antigen-binding region that is a key determinant of antigen specificity (2). On average, mature B-cell subsets express antibodies that have shorter and more negatively charged CDR3s, which is the result of selection against autoreactive and polyreactive B cells (3,4).Each recombined antibody heavy-chain gene is composed of a variable (V H ), diversity (D), and joining (J H ) gene segment. Because the CDR3 region spans the V H -D-J H joint, investigators have asked whether selection might favor B cells whose antibodies use specific V H , D, or J H gene segments. Selection in favor of specific gene segments during B-cell maturation might help to explain the observed maturation-associated changes in CDR3 length and charge, and might suggest a preference for "hard-wired" antigen specificities. Evidence against selection would suggest that differences in CDR3 result exclusively from the nontemplated addition and deletion of nucleotides at the V H -D and D-J H junctions, and therefore that the death of B cells with counterselected CDR3s during maturation is simply the evolutionary cost (3) of maintaining this mechanism of generating antibody diversity.Nearly two decades ago, a low-throughput sequencing study in mice suggested that specific V H gene segments were used at different frequencies by pre-B cells (in which heavy-chain recombination has been completed) and mature B cells in the spleen (5). This observation was interpreted as selection for hard-wired specificities. However, the statistical robustness of this observation was limited by the small number of recombined genes that were sequenced, and although subsequent investigations have detected differences in V H use between pre-B and upstream pro-B cells, they have failed to confirm such differences between pre-B and...