Conformational Flexibility in Respiratory Syncytial Virus G Neutralizing Epitopes.

Respiratory syncytial virus (RSV) is a top cause of severe lower respiratory tract disease and mortality in infants and the elderly. Currently, no vaccine or effective treatment exists for RSV. The RSV G glycoprotein mediates viral attachment to cells and contributes to pathogenesis by modulating host immunity through interactions with the human chemokine receptor CX3CR1. Antibodies targeting the RSV G central conserved domain are protective in both prophylactic and postinfection animal models. Here, we describe the crystal structure of the broadly neutralizing human monoclonal antibody 3G12 bound to the RSV G central conserved domain. Antibody 3G12 binds to a conformational epitope composed of highly conserved residues, explaining its broad neutralization activity. Surprisingly, RSV G complexed with 3G12 adopts a distinct conformation not observed in previously described RSV G-antibody structures. Comparison to other structures reveals that the RSV G central conserved domain is flexible and can adopt multiple conformations in the regions flanking the cysteine noose. We also show that restriction of RSV G flexibility with a proline mutation abolishes binding to antibody 3G12 but not antibody 3D3, which recognizes a different conformation of RSV G. Our studies provide new insights for rational vaccine design, indicating the importance of preserving both the global structural integrity of antigens and local conformational flexibility at antigenic sites, which may elicit a more diverse antibody response and broader protection against infection and disease.IMPORTANCE Respiratory syncytial virus (RSV) causes severe respiratory infections in infants, young children, and the elderly, and currently, no licensed vaccine exists. In this study, we describe the crystal structure of the RSV surface glycoprotein G in complex with a broadly neutralizing human monoclonal antibody. The antibody binds to RSV G at a highly conserved region stabilized by two disulfide bonds, but it captures RSV G in a conformation not previously observed, revealing that this region is both structured and flexible. Importantly, our findings provide insight for the design of vaccines that elicit diverse antibodies, which may provide broad protection from infection and disease.

Structures of respiratory syncytial virus G antigen bound to broadly neutralizing antibodies.

Respiratory syncytial virus (RSV) is a top cause of severe lower respiratory tract disease and mortality in young children and the elderly. The viral envelope G glycoprotein contributes to pathogenesis through its roles in host cell attachment and modulation of host immunity. Although the G glycoprotein is a target of protective RSV-neutralizing antibodies, its development as a vaccine antigen has been hindered by its heterogeneous glycosylation and sequence variability outside a conserved central domain (CCD). We describe the cocrystal structures of two high-affinity broadly neutralizing human monoclonal antibodies bound to the RSV G CCD. The antibodies bind to neighboring conformational epitopes, which we named antigenic sites γ1 and γ2, that span a highly conserved surface, illuminating an important region of vulnerability. We further show that isolated RSV G CCD activates the chemokine receptor CX3CR1 and that antibodies block this activity. These studies provide a template for rational vaccine design targeting this key contributor to RSV disease.

8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme.

8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew-Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar-phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein-DNA interface.

Gelsolin-like activation of villin: calcium sensitivity of the long helix in domain 6.

Villin is a gelsolin-like cytoskeleton regulator localized in the brush border at the apical end of epithelial cells. Villin regulates microvilli by bundling F-actin at low calcium levels and severing it at high calcium levels. The villin polypeptide consists of six gelsolin-like repeats (V1-V6) and the unique, actin binding C-terminal headpiece domain (HP). Villin modular fragment V6-HP requires calcium to stay monomeric and bundle F-actin. Our data show that isolated V6 is monomeric and does not bind F-actin at any level of calcium. We propose that the 40-residue unfolded V6-to-HP linker can be a key regulatory element in villin's functions such as its interactions with F-actin. Here we report a calcium-bound solution nuclear magnetic resonance (NMR) structure of V6, which has a gelsolin-like fold with the long α-helix in the extended conformation. Intrinsic tryptophan fluorescence quenching reveals two-Kd calcium binding in V6 (Kd1 of 22 µM and Kd2 of 2.8 mM). According to our NMR data, the conformation of V6 responds the most to micromolar calcium. We show that the long α-helix and the adjacent residues form the calcium-sensitive elements in V6. These observations are consistent with the calcium activation of F-actin severing by villin analogous to the gelsolin helix-straightening mechanism.

An N-terminal, 830 residues intrinsically disordered region of the cytoskeleton-regulatory protein supervillin contains Myosin II- and F-actin-binding sites.

Supervillin, the largest member of the villin/gelsolin family, is a cytoskeleton regulating, peripheral membrane protein. Supervillin increases cell motility and promotes invasive activity in tumors. Major cytoskeletal interactors, including filamentous actin and myosin II, bind within the unique supervillin amino terminus, amino acids 1-830. The structural features of this key region of the supervillin polypeptide are unknown. Here, we utilize circular dichroism and bioinformatics sequence analysis to demonstrate that the N-terminal part of supervillin forms an extended intrinsically disordered region (IDR). Our combined data indicate that the N-terminus of human and bovine supervillin sequences (positions 1-830) represents an IDR, which is the largest IDR known to date in the villin/gelsolin family. Moreover, this result suggests a potentially novel mechanism of regulation of myosin II and F-actin via the intrinsically disordered N-terminal region of hub protein supervillin.