Structures of Preferred Human IgV Genes-Based Protective Antibodies Identify How Conserved Residues Contact Diverse Antigens and Assign Source of Specificity to CDR3 Loop Variation.

The human Ab response to certain pathogens is oligoclonal, with preferred IgV genes being used more frequently than others. A pair of such preferred genes, IGVK3-11 and IGVH3-30, contributes to the generation of protective Abs directed against the 23F serotype of the pneumonococcal capsular polysaccharide of Streptococcus pneumoniae and against the AD-2S1 peptide of the gB membrane protein of human CMV. Structural analyses of Fab fragments of mAbs 023.102 and pn132p2C05 in complex with portions of the 23F polysaccharide revealed five germline-encoded residues in contact with the key component, l-rhamnose. In the case of the AD-2S1 peptide, the KE5 Fab fragment complex identified nine germline-encoded contact residues. Two of these germline-encoded residues, Arg91L and Trp94L, contact both the l-rhamnose and the AD-2S1 peptide. Comparison of the respective paratopes that bind to carbohydrate and protein reveals that stochastic diversity in both CDR3 loops alone almost exclusively accounts for their divergent specificity. Combined evolutionary pressure by human CMV and the 23F serotype of S. pneumoniae acted on the IGVK3-11 and IGVH3-30 genes as demonstrated by the multiple germline-encoded amino acids that contact both l-rhamnose and AD-2S1 peptide.

Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design.

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.

Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus.

Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

An exomoon survey of 70 cool giant exoplanets and the new candidate Kepler-1708 b-i.

Exomoons represent a crucial missing puzzle piece in our efforts to understand extrasolar planetary systems. To address this deficiency, we here describe an exomoon survey of 70 cool, giant transiting exoplanet candidates found by Kepler. We identify only one exhibiting a moon-like signal that passes a battery of vetting tests: Kepler-1708 b. We show that Kepler-1708 b is a statistically validated Jupiter-sized planet orbiting a Sun-like quiescent star at 1.6 au. The signal of the exomoon candidate, Kepler-1708 b-i, is a 4.8σ effect and is persistent across different instrumental detrending methods, with a 1% false-positive probability via injection-recovery. Kepler-1708 b-i is ~2.6 Earth radii and is located in an approximately coplanar orbit at ~12 planetary radii from its ~1.6 au Jupiter-sized host. Future observations will be necessary to validate or reject the candidate.

Crystallographic definition of the epitope promiscuity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5: vaccine design implications.

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab'. A variety of amino acid substitutions outside the 664DKW666 core epitope are tolerated. However, changes at the 664DKW666 motif itself are restricted to those residues that preserve the aspartate's negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.

ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores.

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

Biological Evaluation and X-ray Co-crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase, an Enzyme from the Redox Metabolism of Trypanosoma.

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (Ki ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site.

The mechanism of GM-CSF inhibition by human GM-CSF auto-antibodies suggests novel therapeutic opportunities.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that can stimulate a variety of cells, but its overexpression leads to excessive production and activation of granulocytes and macrophages with many pathogenic effects. This cytokine is a therapeutic target in inflammatory diseases, and several anti-GM-CSF antibodies have advanced to Phase 2 clinical trials in patients with such diseases, e.g., rheumatoid arthritis. GM-CSF is also an essential factor in preventing pulmonary alveolar proteinosis (PAP), a disease associated with GM-CSF malfunction arising most typically through the presence of GM-CSF neutralizing auto-antibodies. Understanding the mechanism of action for neutralizing antibodies that target GM-CSF is important for improving their specificity and affinity as therapeutics and, conversely, in devising strategies to reduce the effects of GM-CSF auto-antibodies in PAP. We have solved the crystal structures of human GM-CSF bound to antigen-binding fragments of two neutralizing antibodies, the human auto-antibody F1 and the mouse monoclonal antibody 4D4. Coordinates and structure factors of the crystal structures of the GM-CSF:F1 Fab and the GM-CSF:4D4 Fab complexes have been deposited in the RCSB Protein Data Bank under the accession numbers 6BFQ and 6BFS, respectively. The structures show that these antibodies bind to mutually exclusive epitopes on GM-CSF; however, both prevent the cytokine from interacting with its alpha receptor subunit and hence prevent receptor activation. Importantly, identification of the F1 epitope together with functional analyses highlighted modifications to GM-CSF that would abolish auto-antibody recognition whilst retaining GM-CSF function. These results provide a framework for developing novel GM-CSF molecules for PAP treatment and for optimizing current anti-GM-CSF antibodies for use in treating inflammatory disorders.

PLANETARY CANDIDATES OBSERVED BY Kepler. VIII. A FULLY AUTOMATED CATALOG WITH MEASURED COMPLETENESS AND RELIABILITY BASED ON DATA RELEASE 25.

We present the Kepler Object of Interest (KOI) catalog of transiting exoplanets based on searching four years of Kepler time series photometry (Data Release 25, Q1-Q17). The catalog contains 8054 KOIs of which 4034 are planet candidates with periods between 0.25 and 632 days. Of these candidates, 219 are new in this catalog and include two new candidates in multi-planet systems (KOI-82.06 and KOI-2926.05), and ten new high-reliability, terrestrial-size, habitable zone candidates. This catalog was created using a tool called the Robovetter which automatically vets the DR25 Threshold Crossing Events (TCEs) found by the Kepler Pipeline (Twicken et al. 2016). Because of this automation, we were also able to vet simulated data sets and therefore measure how well the Robovetter separates those TCEs caused by noise from those caused by low signal-to-noise transits. Because of these measurements we fully expect that this catalog can be used to accurately calculate the frequency of planets out to Kepler's detection limit, which includes temperate, super-Earth size planets around GK dwarf stars in our Galaxy. This paper discusses the Robovetter and the metrics it uses to decide which TCEs are called planet candidates in the DR25 KOI catalog. We also discuss the simulated transits, simulated systematic noise, and simulated astrophysical false positives created in order to characterize the properties of the final catalog. For orbital periods less than 100 d the Robovetter completeness (the fraction of simulated transits that are determined to be planet candidates) across all observed stars is greater than 85%. For the same period range, the catalog reliability (the fraction of candidates that are not due to instrumental or stellar noise) is greater than 98%. However, for low signal-to-noise candidates found between 200 and 500 days, our measurements indicate that the Robovetter is 73.5% complete and 37.2% reliable across all searched stars (or 76.7% complete and 50.5% reliable when considering just the FGK dwarf stars). We describe how the measured completeness and reliability varies with period, signal-to-noise, number of transits, and stellar type. Also, we discuss a value called the disposition score which provides an easy way to select a more reliable, albeit less complete, sample of candidates. The entire KOI catalog, the transit fits using Markov chain Monte Carlo methods, and all of the simulated data used to characterize this catalog are available at the NASA Exoplanet Archive.

A cell line (HEW) from embryos of haddock (Melanogrammus aeglefinius) and its capacity to tolerate environmental extremes.

Cell lines can be useful experimental tools for studying marine fish, which are often difficult to routinely obtain and maintain in the laboratory. As few cell lines are available from coldwater marine fish, cultures were initiated from late gastrula embryos of haddock (Melanogrammus aeglefinus) in Leibovitz's L-15 with fetal bovine serum (FBS). From one culture, a cell line (HEW) emerged that has been grown for close to 100 population doublings, was heteroploid, and expressed telomerase activity, all of which suggest HEW is immortal. Growth occurred only if FBS was present and was optimal at 12 to 18 degrees C. Usually most cells had an epithelial-like morphology, but under some conditions, cells drew up into round central bodies from which radiated cytoplasmic extensions with multiple branches. These neural-like cells appeared within a few hours of cultures being placed at 28 degrees C or being switch to a simple salt solution (SSS). At 28 degrees C, cells died within 24 h. In SSS, HEW cells survived as a monolayer for at least 7 days. The sensitivity of HEW cells to morphological change and their capacity to withstand starvation should make them useful for investigating cellular responses to environmental stresses.

A point mutation in the Ch3 domain of human IgG3 inhibits antibody secretion without affecting antigen specificity.

Immunoglobulins (Ig) require correct folding and assembly of both heavy (H) and light (L) chains to form a functional H2L2 dimer that is secreted from plasma cells. This process is dependent upon the endoplasmic reticulum (ER) chaperone BiP, which targets improperly, folded or assembled Ig molecules for degradation. While investigating the mechanism of low IgG3 secretion, we identified a missense mutation L368P in the Ch3 region of the human gamma3 H-chain that was associated with impaired secretion of intact and functional Ig. The non-secreted H-chains displayed slower electrophoretic migration than secreted H-chains, consistent with them being glycosylated in the ER but not fully processed in the golgi apparatus and secretory pathway. Reversion of the mutated codon to wild type restored secretion of the IgG3, which displayed the same fine specificity for antigen as non-secreted IgG3. However, the non-secreted IgG3 was not opsonic in an in vitro phagocytosis assay. The results indicate that correct IgG3 Ch3 domain folding is essential for secretion and effective function but does not affect specificity for antigen.

Development and Characterization of Potent Cyclic Acyldepsipeptide Analogues with Increased Antimicrobial Activity.

The problem of antibiotic resistance has prompted the search for new antibiotics with novel mechanisms of action. Analogues of the A54556 cyclic acyldepsipeptides (ADEPs) represent an attractive class of antimicrobial agents that act through dysregulation of caseinolytic protease (ClpP). Previous studies have shown that ADEPs are active against Gram-positive bacteria (e.g., MRSA, VRE, PRSP (penicillin-resistant Streptococcus pneumoniae)); however, there are currently few studies examining Gram-negative bacteria. In this study, the synthesis and biological evaluation of 14 novel ADEPs against a variety of pathogenic Gram-negative and Gram-positive organisms is outlined. Optimization of the macrocyclic core residues and N-acyl side chain culminated in the development of 26, which shows potent activity against the Gram-negative species Neisseria meningitidis and Neisseria gonorrheae and improved activity against the Gram-positive organisms Staphylococcus aureus and Enterococcus faecalis in comparison with known analogues. In addition, the co-crystal structure of an ADEP-ClpP complex derived from N. meningitidis was solved.

Binding to large enzyme pockets: small-molecule inhibitors of trypanothione reductase.

The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure-based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small-molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so-called "mepacrine binding site". The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi.

Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope.

Computational grafting of functional motifs onto scaffold proteins is a promising way to engineer novel proteins with pre-specified functionalities. Typically, protein grafting involves the transplantation of protein side chains from a functional motif onto structurally homologous regions of scaffold proteins. Using this approach, we previously transplanted the human immunodeficiency virus 2F5 and 4E10 epitopes onto heterologous proteins to design novel "epitope-scaffold" antigens. However, side-chain grafting is limited by the availability of scaffolds with compatible backbone for a given epitope structure and offers no route to modify backbone structure to improve mimicry or binding affinity. To address this, we report here a new and more aggressive computational method-backbone grafting of linear motifs-that transplants the backbone and side chains of linear functional motifs onto scaffold proteins. To test this method, we first used side-chain grafting to design new 2F5 epitope scaffolds with improved biophysical characteristics. We then independently transplanted the 2F5 epitope onto three of the same parent scaffolds using the newly developed backbone grafting procedure. Crystal structures of side-chain and backbone grafting designs showed close agreement with both the computational models and the desired epitope structure. In two cases, backbone grafting scaffolds bound antibody 2F5 with 30- and 9-fold higher affinity than corresponding side-chain grafting designs. These results demonstrate that flexible backbone methods for epitope grafting can significantly improve binding affinities over those achieved by fixed backbone methods alone. Backbone grafting of linear motifs is a general method to transplant functional motifs when backbone remodeling of the target scaffold is necessary.

Kepler-36: a pair of planets with neighboring orbits and dissimilar densities.

In the solar system, the planets' compositions vary with orbital distance, with rocky planets in close orbits and lower-density gas giants in wider orbits. The detection of close-in giant planets around other stars was the first clue that this pattern is not universal and that planets' orbits can change substantially after their formation. Here, we report another violation of the orbit-composition pattern: two planets orbiting the same star with orbital distances differing by only 10% and densities differing by a factor of 8. One planet is likely a rocky "super-Earth," whereas the other is more akin to Neptune. These planets are 20 times more closely spaced and have a larger density contrast than any adjacent pair of planets in the solar system.

Structural details of HIV-1 recognition by the broadly neutralizing monoclonal antibody 2F5: epitope conformation, antigen-recognition loop mobility, and anion-binding site.

2F5 is a monoclonal antibody with potent and broadly neutralizing activity against HIV-1. It targets the membrane-proximal external region (MPER) of the gp41 subunit of the envelope glycoprotein and interferes with the process of fusion between viral and host cell membranes. This study presents eight 2F5 F(ab)' crystal structures in complex with various gp41 peptide epitopes. These structures reveal several key features of this antibody-antigen interaction. (1) Whenever free of contacts caused by crystal artifacts, the extended complementarity-determining region H3 loop is mobile; this is true for ligand-free and epitope-bound forms. (2) The interaction between the antibody and the gp41 ELDKWA epitope core is absolutely critical, and there are also close and specific contacts with residues located N-terminal to the epitope core. (3) Residues located at the C-terminus of the gp41 ELDKWA core do not interact as tightly with the antibody. However, in the presence of a larger peptide containing the gp41 fusion peptide segment, these residues adopt a conformation consistent with the start of an alpha-helix. (4) At high sulfate concentrations, the electron density maps of 2F5 F(ab)'-peptide complexes contain a peak that may mark a binding site for phosphate groups of negatively charged lipid headgroups. The refined atomic-level details of 2F5 paratope-epitope interactions revealed here should contribute to a better understanding of the mechanism of 2F5-based virus neutralization, in general, and prove important for the design of potential vaccine candidates intended to elicit 2F5-like antibody production.

Crystal structure of the complex between the F(ab)' fragment of the cross-neutralizing anti-HIV-1 antibody 2F5 and the F(ab) fragment of its anti-idiotypic antibody 3H6.

The monoclonal antibody 2F5 neutralizes a broad range of human immunodeficiency virus-1 isolates via a conserved epitope on the viral glycoprotein gp41. The conformation of the principal epitope is a type I beta-turn centered on gp41 residues (664)DKW(666); in addition, binding studies indicate that residues N- and C-terminal to this core as well as structurally more distant parts of gp41 also contribute to the interaction. Ab2/3H6 is an anti-idiotypic antibody that inhibits the interaction between 2F5 and gp41 and as such, Ab2/3H6 may, in principle, possess a paratope that mimics the gp41 epitope. To establish the potential of Ab2/3H6 to serve as a guide for the design of vaccine components against human immunodeficiency virus, we investigated the crystal structure of the heterodimeric complex of Ab2/3H6 F(ab) and 2F5 F(ab)'. Ab2/3H6 F(ab) binds to 2F5 F(ab)' via a helix-like protrusion formed by residues (58(H))RYSPSLNTRL(67(H)) of the 2F5 F(ab)' variable domain and proximal to but not overlapping with the gp41 (664)DKW(666) epitope-binding pocket. This defines Ab2/3H6 as an anti-idiotypic antibody of the Ab2gamma class, i.e., an antigen-inhibitable idiotype that does not carry the internal image of the linear primary gp41 (662)ELDKWAS(668) epitope.

Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity.

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.