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2.  Two Classes of Broadly Neutralizing Antibodies within a Single Lineage Directed to the High-Mannose Patch of HIV Envelope 
Journal of Virology  2014;89(2):1105-1118.
ABSTRACT
The high-mannose patch of human immunodeficiency virus (HIV) envelope (Env) elicits broadly neutralizing antibodies (bnAbs) during natural infection relatively frequently, and consequently, this region has become a major target of vaccine design. However, it has also become clear that antibody recognition of the region is complex due, at least in part, to variability in neighboring loops and glycans critical to the epitopes. bnAbs against this region have some shared features and some distinguishing features that are crucial to understand in order to design optimal immunogens that can induce different classes of bnAbs against this region. Here, we compare two branches of a single antibody lineage, in which all members recognize the high-mannose patch. One branch (prototype bnAb PGT128) has a 6-amino-acid insertion in CDRH2 that is crucial for broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on gp120, and somatic hypermutation is required to accommodate the neighboring V1 loop glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing viruses with an alternate N334 glycan site but are less able to accommodate glycan heterogeneity. We identify a new somatic variant within this branch that is predominantly dependent on N334. The crystal structure of PGT130 offers insight into differences from PGT128. We conclude that different immunogens may be required to elicit bnAbs that have the optimal characteristics of the two branches of the lineage described.
IMPORTANCE Development of an HIV vaccine is of vital importance for prevention of new infections, and it is thought that elicitation of HIV bnAbs will be an important component of an effective vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV envelope glycoprotein, gp120, are being highlighted as important templates for vaccine design. In particular, bnAbs from IAVI donor 36 (PGT125 to PGT131) have been shown to be extremely broad and potent. Combination of these bnAbs enhanced neutralization breadth considerably, suggesting that an optimal immunogen should elicit several antibodies from this family. Here we study the evolution of this antibody family to inform immunogen design. We identify two classes of bnAbs that differ in their recognition of the high-mannose patch and show that different immunogens may be required to elicit these different classes.
doi:10.1128/JVI.02905-14
PMCID: PMC4300629  PMID: 25378488
3.  CD4-induced activation in a soluble HIV-1 Env trimer 
The HIV envelope glycoprotein (Env) trimer undergoes receptor-induced conformational changes that drive fusion of the viral and cellular membranes. Env conformational changes have been observed using low-resolution electron microscopy, but only large-scale rearrangements have been visible. Here, we use Hydrogen/Deuterium-exchange and oxidative labeling to gain a more precise understanding of the unliganded and CD4-bound forms of soluble Env trimers (SOSIP.664), including their glycan composition. CD4 activation induces reorganization of bridging sheet elements, V1/V2 and V3, much of the gp120 inner domain, and the gp41 fusion subunit. Two CD4 binding site-targeted inhibitors have substantially different effects: NBD-556 partially mimics CD4-induced destabilization of the V1/V2 and V3 crown, while BMS-806 only affects regions around the gp120/gp41 interface. The structural information presented here increases our knowledge of CD4- and small molecule-induced conformational changes in Env and the allosteric pathways that lead to membrane fusion.
doi:10.1016/j.str.2014.05.001
PMCID: PMC4231881  PMID: 24931470
Env trimers; glycoprotein; glycoform; SOSIP; gp140; HD exchange; deuterium exchange; oxidative labeling; CD4; BMS-806; NBD-556
4.  A structural analysis of M protein in coronavirus assembly and morphology 
Journal of structural biology  2010;174(1):11-22.
The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy, tomography and statistical analysis. We present evidence that suggests M can adopt two conformations and that membrane curvature is regulated by one M conformer. Elongated M protein is associated with rigidity, clusters of spikes and a relatively narrow range of membrane curvature. In contrast, compact M protein is associated with flexibility and low spike density. Analysis of several types of virus-like particles and virions revealed that S protein, N protein and genomic RNA each help to regulate virion size and variation, presumably through interactions with M. These findings provide insight into how M protein functions to promote virus assembly.
doi:10.1016/j.jsb.2010.11.021
PMCID: PMC4486061  PMID: 21130884
Cryo-electron microscopy; cryo-electron tomography; pleomorphic virus structure; coronavirus; viral matrix protein
5.  Crystal structure of a putative quorum sensing-regulated protein (PA3611) from the Pseudomonas-specific DUF4146 family 
Proteins  2013;82(6):1086-1092.
Pseudomonas aeruginosa is an opportunistic pathogen commonly found in humans and other organisms and is an important cause of infection, especially in patients with compromised immune defense mechanisms. The PA3611 gene of P. aeruginosa PAO1 encodes a secreted protein of unknown function, which has been recently classified into a small Pseudomonas-specific protein family called DUF4146. As part of our effort to extend structural coverage of novel protein space and provide a structure-based functional insight into new protein families, we report the crystal structure of PA3611, the first structural representative of the DUF4146 protein family.
doi:10.1002/prot.24455
PMCID: PMC4006323  PMID: 24174223
Pseudomonas-specific protein family; DUF4146; Pfam PF13652; virulence factor; quorum-sensing; JCSG; structural genomics
6.  Characterization of a Broadly Neutralizing Monoclonal Antibody That Targets the Fusion Domain of Group 2 Influenza A Virus Hemagglutinin 
Journal of Virology  2014;88(23):13580-13592.
ABSTRACT
Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043.
IMPORTANCE The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.
doi:10.1128/JVI.02289-14
PMCID: PMC4248980  PMID: 25210195
7.  Stapled HIV-1 Peptides Recapitulate Antigenic Structures and Engage Broadly Neutralizing Antibodies 
Nature structural & molecular biology  2014;21(12):1058-1067.
Hydrocarbon stapling can restore bioactive, α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here, we explore the capacity of peptide stapling to generate high fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease-resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically-stabilized antigens for vaccination.
doi:10.1038/nsmb.2922
PMCID: PMC4304871  PMID: 25420104
8.  Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers 
Immunity  2014;40(5):669-680.
Summary
All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. As PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer.
doi:10.1016/j.immuni.2014.04.008
PMCID: PMC4057017  PMID: 24768348
9.  Broadly neutralizing HIV antibodies define a glycan-dependent epitope on the pre-fusion conformation of the gp41 protein on cleaved Envelope trimers 
Immunity  2014;40(5):657-668.
SUMMARY
Broadly neutralizing antibodies to HIV are much sought-after (a) to guide vaccine design, both as templates and to inform on the authenticity of vaccine candidates, (b) to assist in structural studies and (c) as potential therapeutics. However, the number of targets on the viral envelope spike for such antibodies is limited. Here, we describe a set of human monoclonal antibodies that define a previously undefined target on HIV Env. The antibodies recognize a glycan-dependent epitope on the prefusion conformation of gp41 and unambiguously distinguish cleaved from uncleaved Env trimers, an important property given increasing evidence that cleavage is required for vaccine candidates that seek to mimic the functional HIV envelope spike. The availability of this set of antibodies expands the number of vaccine targets on HIV and provides reagents to characterize the native envelope spike.
doi:10.1016/j.immuni.2014.04.009
PMCID: PMC4070425  PMID: 24768347
10.  Extreme-Depth Re-sequencing of Mitochondrial DNA Finds No Evidence of Paternal Transmission in Humans 
PLoS Genetics  2015;11(5):e1005040.
Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level.
Author Summary
Emerging evidence raises the possibility that human mitochondrial DNA (mtDNA) is not strictly maternally inherited, but it has not been technically possible to test this hypothesis directly. We identified trios with discordant mtDNA haplotypes, parent-offspring trios were validated using polymorphic microsatellites, and then used extreme-high depth mtDNA re-sequencing to look for paternally transmitted mtDNA. Despite having up to ~1.2 million-fold coverage of mtDNA, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans. Our findings exclude a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals.
doi:10.1371/journal.pgen.1005040
PMCID: PMC4431825  PMID: 25973765
11.  DNA Methylation Is Globally Disrupted and Associated with Expression Changes in Chronic Obstructive Pulmonary Disease Small Airways 
DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illumina’s Infinium HM27 and Affymetrix’s Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2–related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.
doi:10.1165/rcmb.2013-0304OC
PMCID: PMC4068945  PMID: 24298892
chronic obstructive pulmonary disease; small airways; epigenetic regulation; DNA methylation; integrative omics
12.  Fluorogenic Small Molecules Requiring Reaction with a Specific Protein to Create a Fluorescent Conjugate for Biological Imaging–What we Know and What we Need to Learn 
Biopolymers  2014;101(5):484-495.
We seek fluorogenic small molecules that generate a fluorescent conjugate signal if and only if they react with a given protein-of-interest (i.e., small molecules for which non-covalent binding to the protein-of-interest is insufficient to generate fluorescence). Consequently, it is the new chemical entity afforded by the generally irreversible reaction between the small molecule and the protein-of-interest that enables the energy of an electron occupying the lowest unoccupied molecular orbital (LUMO) of the chromophore to be given off as a photon instead of being dissipated by non-radiative mechanisms in complex biological environments. This category of fluorogenic small molecules is created by starting with environmentally sensitive fluorophores that are modified by an essential functional group that efficiently quenches the fluorescence until a chemoselective reaction between that functional group and the protein-of-interest occurs, yielding the fluorescent conjugate. Fluorogenic small molecules are envisioned to be useful for a wide variety of applications, including live cell imaging without the requirement for washing steps and pulse-chase kinetic analyses of protein synthesis, trafficking, degradation, etc.
doi:10.1002/bip.22407
PMCID: PMC4128284  PMID: 24105107
13.  Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies 
Nature  2014;509(7498):55-62.
Summary
Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from CAPRISA-donor CAP256; each antibody contained the protruding tyrosine-sulfated, anionic antigen-binding loop (CDR H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30–38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation, an important vaccine insight.
doi:10.1038/nature13036
PMCID: PMC4395007  PMID: 24590074
14.  Receptor mimicry by antibody F045-092 facilitates universal binding to the H3 subtype of influenza virus 
Nature communications  2014;5:3614.
Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012-2013. Here, we describe an antibody, F045-092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045-092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045-092 extends its recognition to divergent subtypes, including H1, H2, and H13, using the enhanced avidity of its IgG to overcome lower affinity Fab binding, as observed with other receptor-binding site antibodies. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small molecule therapeutics.
doi:10.1038/ncomms4614
PMCID: PMC4358779  PMID: 24717798
15.  Crystal Structure of a Two-Subunit TrkA Octameric Gating Ring Assembly 
PLoS ONE  2015;10(3):e0122512.
The TM1088 locus of T. maritima codes for two proteins designated TM1088A and TM1088B, which combine to form the cytosolic portion of a putative Trk K+ transporter. We report the crystal structure of this assembly to a resolution of 3.45 Å. The high resolution crystal structures of the components of the assembly, TM1088A and TM1088B, were also determined independently to 1.50 Å and 1.55 Å, respectively. The TM1088 proteins are structurally homologous to each other and to other K+ transporter proteins, such as TrkA. These proteins form a cytosolic gating ring assembly that controls the flow of K+ ions across the membrane. TM1088 represents the first structure of a two-subunit Trk assembly. Despite the atypical genetics and chain organization of the TM1088 assembly, it shares significant structural homology and an overall quaternary organization with other single-subunit K+ gating ring assemblies. This structure provides the first structural insights into what may be an evolutionary ancestor of more modern single-subunit K+ gating ring assemblies.
doi:10.1371/journal.pone.0122512
PMCID: PMC4380455  PMID: 25826626
16.  Comprehensive Antigenic Map of a Cleaved Soluble HIV-1 Envelope Trimer 
PLoS Pathogens  2015;11(3):e1004767.
The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.
Author Summary
The discovery of new broadly neutralizing antibodies against various epitopes on the HIV-1 envelope glycoprotein trimer and increased knowledge of its structure are guiding vaccine design. To increase our understanding of the interrelationships among the different epitopes, we generated a detailed antigenic map of the trimer using a variety of techniques. We have uncovered various mechanisms whereby antibodies can influence each other’s binding. The resulting antigenic map should further aid in design of HIV-1 vaccines to induce broadly neutralizing antibodies and in devising cocktails of such antibodies for therapeutic use.
doi:10.1371/journal.ppat.1004767
PMCID: PMC4373910  PMID: 25807248
17.  Structure of 2G12 Fab2 in Complex with Soluble and Fully Glycosylated HIV-1 Env by Negative-Stain Single-Particle Electron Microscopy 
Journal of Virology  2014;88(17):10177-10188.
ABSTRACT
The neutralizing anti-HIV-1 antibody 2G12 is of particular interest due to the sterilizing protection it provides from viral challenge in animal models. 2G12 is a unique, domain-exchanged antibody that binds exclusively to conserved N-linked glycans that form the high-mannose patch on the gp120 outer domain centered on a glycan at position N332. Several glycans in and around the 2G12 epitope have been shown to interact with other potent, broadly neutralizing antibodies; therefore, this region constitutes a supersite of vulnerability on gp120. While crystal structures of 2G12 and 2G12 bound to high-mannose glycans have been solved, no structural information that describes the interaction of 2G12 with gp120 or the Env trimer is available. Here, we present a negative-stain single-particle electron microscopy reconstruction of 2G12 Fab2 in complex with a soluble, trimeric Env at ∼17-Å resolution that reveals the antibody's interaction with its native and fully glycosylated epitope. We also mapped relevant glycans in this epitope by fitting high-resolution crystal structures and by performing neutralization assays of glycan knockouts. In addition, a reconstruction at ∼26 Å of the ternary complex formed by 2G12 Fab2, soluble CD4, and Env indicates that 2G12 may block membrane fusion by induced steric hindrance upon primary receptor binding, thereby abrogating Env's interaction with coreceptor(s). These structures provide a basis for understanding 2G12 binding and neutralization, and our low-resolution model and glycan assignments provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope.
IMPORTANCE HIV-1 is a human virus that results in the deaths of millions of people around the world each year. While there are several effective therapeutics available to prolong life, a vaccine is the best long-term solution for curbing this global epidemic. Here, we present structural data that reveal the viral binding site of one of the first HIV-1-neutralizing antibodies isolated, 2G12, and provide a rationale for its effectiveness. These structures provide a basis for higher-resolution studies to determine the molecular nature of the 2G12 epitope, which will aid in vaccine design and antibody-based therapies.
doi:10.1128/JVI.01229-14
PMCID: PMC4136306  PMID: 24965454
18.  A Unique Human Mycoplasma Protein that Generically Blocks Antigen-Antibody Union 
Science (New York, N.Y.)  2014;343(6171):656-661.
We report the discovery and crystal structure of a human mycoplasma protein, Protein M, which binds with high affinity to antibodies, predominantly through attachment to the variable region of the κ and λ light chains. Protein M broadly blocks antibody-antigen union and its mechanism of inhibition is of considerable interest because, as a diversity system, the binding mode of each antibody is different. Protein M thus appears to function by a mechanism that is independent of the sequences of members of the extensive antibody repertoire. By anchoring to conserved regions of the antibody light chains, Protein M is in a position to extend its large C-terminal domain over the antibody combining site and block entrance to macromolecular antigens.
doi:10.1126/science.1246135
PMCID: PMC3987992  PMID: 24503852
19.  Structures of a bifunctional cell-wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 dl-endopeptidase 
Journal of molecular biology  2013;426(1):10.1016/j.jmb.2013.09.011.
Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell-wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of two CwlT from pathogens Staphylococcus aureus mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific for tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release, or closing during catalysis.
doi:10.1016/j.jmb.2013.09.011
PMCID: PMC3872209  PMID: 24051416
bifunctional cell-wall lysin; bacterial lysozyme; muramidase; NlpC/P60 endopeptidase; Tn916 family conjugative transposons
20.  The Telehealth Skills, Training, and Implementation Project: An Evaluation Protocol 
JMIR Research Protocols  2015;4(1):e2.
Background
Telehealth appears to be an ideal mechanism for assisting rural patients and doctors and medical students/registrars in accessing specialist services. Telehealth is the use of enhanced broadband technology to provide telemedicine and education over distance. It provides accessible support to rural primary care providers and medical educators. A telehealth consultation is where a patient at a general practice, with the assistance of the general practitioner or practice nurse, undertakes a consultation by videoconference with a specialist located elsewhere. Multiple benefits of telehealth consulting have been reported, particularly those relevant to rural patients and health care providers. However there is a paucity of research on the benefits of telehealth to medical education and learning.
Objective
This protocol explains in depth the process that will be undertaken by a collaborative group of universities and training providers in this unique project.
Methods
Training sessions in telehealth consulting will be provided for participating practices and students. The trial will then use telehealth consulting as a real-patient learning experience for students, general practitioner trainees, general practitioner preceptors, and trainees.
Results
Results will be available when the trial has been completed in 2015.
Conclusions
The protocol has been written to reflect the overarching premise that, by building virtual communities of practice with users of telehealth in medical education, a more sustainable and rigorous model can be developed. The Telehealth Skills Training and Implementation Project will implement and evaluate a theoretically driven model of Internet-facilitated medical education for vertically integrated, community-based learning environments
doi:10.2196/resprot.3613
PMCID: PMC4296098  PMID: 25567780
telehealth; medical education; enhanced broadband
21.  Crystal structure of a member of a novel family of dioxygenases (PF10014) reveals a conserved cupin fold and active site 
Proteins  2013;82(1):164-170.
PF10014 is a novel family of 2-oxyglutarate-Fe2+-dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the β-strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site.
doi:10.1002/prot.24362
PMCID: PMC3920835  PMID: 23852666
PF10014/BsmA; cupin dioxygenase; free amino acids; 2-oxyglutarate; ferrous iron
22.  Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 Env trimer 
Science (New York, N.Y.)  2013;342(6165):1484-1490.
The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo-electron microscopy reconstruction and structural model of a cleaved, soluble SOSIP gp140 trimer in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 Å resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1 and HR2 domains, and shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.
doi:10.1126/science.1245627
PMCID: PMC3954647  PMID: 24179160
23.  Crystal structure of a soluble cleaved HIV-1 envelope trimer 
Science (New York, N.Y.)  2013;342(6165):10.1126/science.1245625.
HIV-1 entry into CD4+ target cells is mediated by cleaved envelope glycoprotein (Env) trimers that have been challenging to characterize structurally. Here, we describe the crystal structure at 4.7 Å of an antigenically near-native, cleaved, stabilized, soluble Env trimer (termed BG505 SOSIP.664 gp140) in complex with a potent broadly neutralizing antibody, PGT122. The structure shows a pre-fusion state of gp41, the interaction between the component gp120 and gp41 subunits, and how a close association between the gp120 V1/V2/V3 loops stabilizes the trimer apex around the three-fold axis. The complete epitope of PGT122 on the trimer involves gp120 V1, V3 and several surrounding glycans. This trimer structure advances our understanding of how Env functions and is presented to the immune system, and provides a blueprint for structure-based vaccine design.
doi:10.1126/science.1245625
PMCID: PMC3886632  PMID: 24179159
24.  Preferential recognition of avian-like receptors in human influenza A H7N9 viruses 
Science (New York, N.Y.)  2013;342(6163):1230-1235.
The 2013 outbreak of avian-origin H7N9 influenza in eastern China has raised concerns about its ability to transmit in the human population. The hemagglutinin glycoprotein of most human H7N9 viruses carries Leu226, a residue linked to adaptation of H2N2 and H3N2 pandemic viruses to human receptors. However, glycan array analysis of the H7 hemagglutinin reveals negligible binding to human-like α2-6-linked receptors and strong preference for a subset of avian-like α2-3-linked glycans recognized by all avian H7 viruses. Crystal structures of H7N9 hemagglutinin and six hemagglutinin-glycan complexes have elucidated the structural basis for preferential recognition of avian-like receptors. These findings suggest that the current human H7N9 viruses are poorly adapted for efficient human-to-human transmission.
doi:10.1126/science.1243761
PMCID: PMC3954636  PMID: 24311689
25.  Alternative Recognition of the Conserved Stem Epitope in Influenza A Virus Hemagglutinin by a VH3-30-Encoded Heterosubtypic Antibody 
Journal of Virology  2014;88(12):7083-7092.
ABSTRACT
A human monoclonal heterosubtypic antibody, MAb 3.1, with its heavy chain encoded by VH3-30, was isolated using phage display with immobilized hemagglutinin (HA) from influenza virus A/Japan/305/1957(H2N2) as the target. Antibody 3.1 potently neutralizes influenza viruses from the H1a clade (i.e., H1, H2, H5, H6) but has little neutralizing activity against the H1b clade. Its crystal structure in complex with HA from a pandemic H1N1 influenza virus, A/South Carolina/1/1918(H1N1), revealed that like other heterosubtypic anti-influenza virus antibodies, MAb 3.1 contacts a hydrophobic groove in the HA stem, primarily using its heavy chain. However, in contrast to the closely related monoclonal antibody (Mab) FI6 that relies heavily on HCDR3 for binding, MAb 3.1 utilizes residues from HCDR1, HCDR3, and framework region 3 (FR3). Interestingly, HCDR1 of MAb 3.1 adopts an α-helical conformation and engages in hydrophobic interactions with the HA very similar to those of the de novo in silico-designed and affinity-matured synthetic protein HB36.3. These findings improve our understanding of the molecular requirements for binding to the conserved epitope in the stem of the HA protein and, therefore, aid the development of more universal influenza vaccines targeting these epitopes.
IMPORTANCE Influenza viruses rapidly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by acquiring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by immunization or infection protect only against the immunizing or closely related strains. Here, we describe a novel monoclonal antibody that recognizes the conserved heterosubtypic epitope in the stem of influenza A virus hemagglutinin. This antibody, referred to as MAb 3.1, recognizes its epitope in a manner that resembles recognition of a similar epitope by the de novo in silico-designed and affinity-matured synthetic protein HB36.3. Thus, besides providing novel insights into the molecular interactions between heterosubtypic antibodies and influenza virus hemagglutinin, MAb 3.1 demonstrates that de novo in silico-designed and affinity-matured synthetic proteins can foretell naturally selected antibody binding. This knowledge will aid development of a pan-influenza virus vaccine.
doi:10.1128/JVI.00178-14
PMCID: PMC4054347  PMID: 24719426

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