Edgar Davidson

Primary structure of the bc1 complex of Rhodopseudomonas capsulata: Nucleotide sequence of the pet operon encoding the Rieske cytochrome b, and cytochrome c1 apoproteins

The nucleotide sequence of the pet operon of Rhodopseudomonas capsulata strain SB1003 has been determined. This operon consists of the petA, petB and petC genes, which encode the Rieske Fe-S protein, cytochrome b and cytochrome c1, respectively, all components of the ubiquinol-cytochrome c2 oxidoreductase. The deduced amino acid sequences of the pet genes show homology to the corresponding proteins from other organisms, and particularly high homologies (over 90% for amino acid and nucleotide sequences) to the previously described fbc operon from a strain previously identified as Rhodopseudomonas spheroides GA. The amino acid sequences of the pet proteins are discussed with reference to the structure and function of the ubiquinol-cytochrome c2 oxidoreductase.

Isolation of the structural genes for the Rieske FeS protein, cytochrome b and cytochrome c1 all components of the ubiquinol: Cytochrome c2 oxidoreductase complex of Rhodopseudomonas capsulata☆

The structural genes for the Rieske Fe-S protein (petA), cytochrome b (petB) and cytochrome c1 (petC) subunits of the ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodopseudomonas capsulata have been cloned by complementation, using a mutant defective in this complex. The location of these genes on the obtained plasmid, pR14A, was determined using synthetic mixed oligonucleotide probes corresponding to highly conserved amino acid sequences of these proteins from various organisms. Their correct identity was established by partial sequencing. The petA, petB and petC genes were found to lie close to each other in this order, spanning two adjacent EcoRI fragments of 2.7 X 10(3) and 1.3 X 10(3) base-pairs, respectively. An insertion-deletion mutation, covering most of petB and all of petC and an insertion mutation, located in petB were constructed in vitro and were introduced into the chromosome of an otherwise wild-type strain by gene transfer agent-mediated genetic crosses. The bc-1 mutants obtained were defective in photosynthesis but, as expected, they could grow by respiration because of a branched respiratory pathway. Therefore, in R. capsulata a functional bc1 complex is essential in vivo for photosynthesis but not for respiration. Further, in the respiratory pathway the branch point must be before the bc1 complex, most likely at the quinone pool. These mutants were also proficient in anaerobic growth in the presence of dimethylsulfoxide, indicating that a functional bc1 complex is not required for this pathway. Several other insertions and deletions, located outside of the pet gene cluster, were also constructed. The ability of these latter mutants to grow photosynthetically suggested that no other gene essential for photosynthesis is located in the proximity of the pet cluster. The plasmid pR14A was shown to complement in trans the bc-1 insertion or insertion-deletion mutants, indicating that the pet genes were expressed in R. capsulata. Cross-hybridization experiments showed that the pet cluster was quite distinct from other known genes involved in photosynthesis.

Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

ABSTRACT Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epitope conservation, and epitope accessibility—also remain largely unknown. To help define how each MAb interacts with GP, here we used comprehensive alanine-scanning mutagenesis (shotgun mutagenesis), neutralization escape, and whole virion binding to define each MAbs specific epitope, epitope accessibility, epitope conservation, and apparent affinity. Each of the six therapeutic MAbs binds nonidentical epitopes in the GP base, glycan cap, or mucin-like domain. Their apparent affinity, epitope complementarity, and epitope accessibility helps explain why MAbs 4G7 and 13C6 are more protective than 2G4 and 1H3. The mucin-like domain MAbs 6D8 and 13F6 bind with the strongest apparent affinity, helping to explain their effectiveness in vivo despite their inability to neutralize virus. IMPORTANCE Ebola virus disease (EVD) can be caused by four different filovirus family members, including Ebola virus (EBOV), which infected 10 times more people in western Africa over the last year than all previous EVD outbreaks combined, with a number of cases distributed across the globe by travelers. Cocktails of inhibitory monoclonal antibodies (MAbs), such as ZMAb, MB-003, and in particular ZMapp, have demonstrated in animal models some of the most significant therapeutic potential for treating EVD, and in 2014, 15 patients were treated with ZMapp or ZMAb under compassionate-use protocols. Here, we have defined the epitope features for the most important therapeutic MAbs against EBOV developed to date. Defining the epitopes and binding characteristics for these MAbs, as well as the commonly used reference MAb KZ52, helps explain their breadth of reactivity against different ebolavirus species, predict viral evasion against these MAbs, and design new cocktails of MAbs with improved complementarity.

A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes

Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their ‘epitopes’, can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high‐throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large‐scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384‐well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein‐coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs.

Rhodobacter capsulatus MT113: a single mutation results in the absence of c-type cytochromes and in the absence of the cytochrome bc1 complex

Abstract MT113, a nonphotosynthetic mutant of Rhodobacter capsulatus previously characterized as lacking cytochrome c2 is shown to lack also cytochrome c1, the Rieske iron-sulfur cluster and the antimycin sensitive semiquinone Q⨪c, all components of the cytochrome bc1 complex. Although MT113 contained b-type cytochromes and other iron-sulfur clusters at nearly wild-type level, it lacks c-type cytochromes. Based on antibody detection, c2 apoprotein was absent in MT113, however the apoproteins corresponding to the cytochromes b and c1 and the Rieske iron-sulfur cluster were present in reduced amounts. Genetic analysis indicated that the lesion appears to be due to a single mutation which is not localized in the structural genes of cytochrome c2 or the bc1 complex. These data taken together suggest that the pleiotropic mutation in MT113 might be related to the biosynthesis of c-type cytochromes.

Atomic-level functional model of dengue virus Envelope protein infectivity

Significance Dengue virus (DENV) infects nearly 400 million people annually, and approximately 40% of the world’s population lives at risk for infection. Without a therapeutic or vaccine available, DENV remains a major public health burden. Our studies provide a comprehensive structure–function analysis of the DENV Envelope protein, integrating information from numerous Envelope structures with our functional data into a cohesive mechanistic model. This model describes the dynamic processes by which specific residue interactions within Envelope mediate infectivity—from fusion-loop triggering to hinge movement to membrane fusion—and may also apply to related class II viral fusion proteins. Because of the importance of the specific amino acids identified, our results also identify new functional residue targets for DENV vaccines and therapeutics. A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through late-stage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity.

Cytochrome c2 mutants of Rhodobacter capsulatus

Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.

Protective Capacity of the Human Anamnestic Antibody Response during Acute Dengue Virus Infection

ABSTRACT Half of the worlds population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo. IMPORTANCE Antibodies can protect from symptomatic dengue virus infection. However, it is not easy to assess which classes of antibodies provide protection because in vitro assays are not always predictive of in vivo protection. During a repeat infection, dengue virus-specific immune memory cells are reactivated and large amounts of antibodies are produced. By studying antibodies cloned from patients with heterologous secondary infection, we tested the protective value of the serotype-cross-reactive “recall” or “anamnestic” response. We found that results from in vitro neutralization assays did not always correlate with the ability of the antibodies to reduce viremia in a mouse model. In addition, a decrease of viremia in mice did not necessarily improve survival. The most protective antibodies were stable at pH 5, suggesting that antibody binding in the endosomes, after the antibody-virus complex is internalized, might be important to block virus spread in the organism.

The Bitter Taste Receptor TAS2R16 Achieves High Specificity and Accommodates Diverse Glycoside Ligands by using a Two-faced Binding Pocket

Although bitter taste receptors (TAS2Rs) are important for human health, little is known of the determinants of ligand specificity. TAS2Rs such as TAS2R16 help define gustatory perception and dietary preferences that ultimately influence human health and disease. Each TAS2R must accommodate a broad diversity of chemical structures while simultaneously achieving high specificity so that diverse bitter toxins can be detected without all foods tasting bitter. However, how these G protein-coupled receptors achieve this balance is poorly understood. Here we used a comprehensive mutation library of human TAS2R16 to map its interactions with existing and novel agonists. We identified 13 TAS2R16 residues that contribute to ligand specificity and 38 residues whose mutation eliminated signal transduction by all ligands, providing a comprehensive assessment of how this GPCR binds and signals. Our data suggest a model in which hydrophobic residues on TM3 and TM7 form a broad ligand-binding pocket that can accommodate the diverse structural features of β-glycoside ligands while still achieving high specificity.

Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles

Significance Generating mAbs against the native extracellular epitopes of multispanning membrane proteins is challenging, and as a result, few nonpeptidic mAbs against transporters have ever been isolated. Our approach here using virus-like particles and divergent host species for immunizations provides a means to overcome these challenges. The specific mAbs isolated here recognize native GLUT4 on the cell surface and can distinguish its different conformational states, thus representing some of the only state-specific mAbs ever isolated against any transporter. Epitope mapping of these mAbs revealed their binding sites as well as the mechanisms by which amino acids control the inward-open and outward-open states of GLUT4. Our studies demonstrate a valuable platform to isolate functional mAbs against important multispanning membrane proteins. The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.