Crystallization and preliminary diffraction studies of native and selenomethionine CcmG (CycY, DsbE)

t Disulfide-bond (Dsb) proteins are a family of redox proteins containing a Cys-X-X-Cys motif. They are essential for disulfide-bond exchange in the bacterial periplasm and are necessary for the correct folding and function of many secreted proteins. CcmG (DsbE) is a reducing Dsb protein required for cytochrome c maturation. Crystals of Bradyrhizobium japonicum CcmG have been obtained that diffract X-rays to 1.14 Angstrom resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell parameters a = 35.1, b = 48.2, c = 90.2 Angstrom. Selenomethionine CcmG was expressed without using a methionine auxotroph or methionine-pathway inhibition and was purified without reducing agents

The Fc region of an antibody impacts the neutralization of West Nile viruses in different maturation states

Flavivirus-infected cells secrete a structurally heterogeneous population of viruses because of an inefficient virion maturation process. Flaviviruses assemble as noninfectious, immature virions composed of trimers of envelope (E) and precursor membrane (prM) protein heterodimers. Cleavage of prM is a required process during virion maturation, although this often remains incomplete for infectious virus particles. Previous work demonstrated that the efficiency of virion maturation could impact antibody neutralization through changes in the accessibility of otherwise cryptic epitopes on the virion. In this study, we show that the neutralization potency of monoclonal antibody (MAb) E33 is sensitive to the maturation state of West Nile virus (WNV), despite its recognition of an accessible epitope, the domain III lateral ridge (DIII-LR). Comprehensive epitope mapping studies with 166 E protein DIII-LR variants revealed that the functional footprint of MAb E33 on the E protein differs subtly from that of the well-characterized DIII-LR MAb E16. Remarkably, aromatic substitutions at E protein residue 306 ablated the maturation state sensitivity of E33 IgG, and the neutralization efficacy of E33 Fab fragments was not affected by changes in the virion maturation state. We propose that E33 IgG binding on mature virions orients the Fc region in a manner that impacts subsequent antibody binding to nearby sites. This Fc-mediated steric constraint is a novel mechanism by which the maturation state of a virion modulates the efficacy of the humoral immune response to flavivirus infection

Structural basis of differential neutralization of DENV-1 genotypes by an antibody that recognizes a cryptic epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC' loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC' loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development

Potent Dengue virus neutralization by a therapeutic antibody with low monovalent affinity requires bivalent engagement

We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface

The Structure, Dynamics and Electronic Structure of Liquid Ag-Se Alloys Investigated by Ab Initio Simulation

Ab initio molecular-dynamics simulations have been used to investigate the structure, dynamics and electronic properties of the liquid alloy Ag(1-x)Se(x) at 1350 K and at the three compositions x=0.33, 0.42 and 0.65. The calculations are based on density-functional theory in the local density approximation and on the pseudopotential plane-wave method. The reliability of the simulations is confirmed by detailed comparisons with very recent neutron diffraction results for the partial structure factors and radial distribution functions (RDF) of the stoichiometric liquid Ag2Se. The simulations show a dramatic change of the Se-Se RDF with increasing Se content. This change is due to the formation of Se clusters bound by covalent bonds, the Se-Se bond length being almost the same as in pure c-Se and l-Se. The clusters are predominantly chain-like, but for higher x a large fraction of 3-fold coordinated Se atoms is also found. It is shown that the equilibrium fractions of Se present as isolated atoms and in clusters can be understood on a simple charge-balance model based on an ionic interpretation. The Ag and Se diffusion coefficients both increase with Se content, in spite of the Se clustering. An analysis of the Se-Se bond dynamics reveals surprisingly short bond lifetimes of less than 1 ps. The changes in the density of states with composition arise directly from the formation of Se-Se covalent bonds. Results for the electronic conductivity obtained using the Kubo-Greenwood approximation are in adequate agreement with experiment for l-Ag2Se, but not for the high Se contents. Possible reasons for this are discussed.Comment: 14 pages, Revtex, 14 Postscript figures embedded in the tex

Structure and relaxations in liquid and amorphous Selenium

We report a molecular dynamics simulation of selenium, described by a three-body interaction. The temperatures T_g and T_c and the structural properties are in agreement with experiment. The mean nearest neighbor coordination number is 2.1. A small pre-peak at about 1 AA^-1 can be explained in terms of void correlations. In the intermediate self-scattering function, i.e. the density fluctuation correlation, classical behavior, alpha- and beta-regimes, is found. We also observe the plateau in the beta-regime below T_g. In a second step, we investigated the heterogeneous and/or homogeneous behavior of the relaxations. At both short and long times the relaxations are homogeneous (or weakly heterogeneous). In the intermediate time scale, lowering the temperature increases the heterogeneity. We connect these different domains to the vibrational (ballistic), beta- and alpha-regimes. We have also shown that the increase in heterogeneity can be understood in terms of relaxations

Development of a highly protective combination monoclonal antibody therapy against Chikungunya virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans

HPCSA Serious Injury Narrative Test guideline

Current South African Road Accident Fund (RAF) legislation requires a medical determination of the seriousness of injuries sustained in motor vehicle accidents to determine whether the claimant is entitled to a claim for general damages. Such medical assessments are submitted in the form of RAF 4 Serious Injury Assessment Reports. Contested claims for serious injury are referred to the Health Professions Council of South Africa (HPCSA) Appeal Tribunals for final determination. The legislation prescribes 2 instruments, namely the American Medical Association (AMA) Guides (6th edition) and the Narrative Test for this purpose. Whereas the AMA Guides are published in a comprehensive book, and training courses are provided in their use, existing legislation does not provide any indication of the required structure, content or criteria of a Narrative Test report. This document is published by the HPCSA Appeal Tribunals as a guideline to the performance of the Narrative Test; what it is, reasons for applying it and who should compile it, as well as the required structure, content and criteria thereof. A Narrative Test Report should include relevant and meaningful comment in relation to each of the 6 sections described in the article.