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The human immune response to an infection by Gram-negative bacteria involves detection of lipopolysaccharides (LPS), also known as endotoxins, which comprise the bacterial outer cell wall. Distinct from mammalian glycolipid structures, LPS have a conserved chemical pattern that is recognized by the pattern recognition receptor complex formed by myeloid differentiation protein 2 (MD-2) and toll-like receptor 4 (TLR4). A remarkable immune-mediated structure-toxicity relationship has been defined that relates to the number of acyl chains in the endotoxin. While there is a clear correlation between endotoxin acylation and elicited agonist or antagonist responses, the 3D structural basis of this relationship remains unclear. In order to explore, at atomic-resolution, the effects of a range of chemically distinct endotoxins on the structure and dynamics of their MD-2·endotoxin complexes, we examined a series of variably acylated lipid A molecules from Escherichia coli and Neisseria meningitidis in complex with human MD-2. Through the application of molecular dynamics simulations, in concert with experimental data, we have identified specific structural and dynamic features of the MD-2-endotoxin complexes that may control dimerization of TLR4 molecules. As dimerization is central to the release of downstream chemical mediators, the results provide a structural foundation for the ability of endotoxins to act as either agonists or antagonists of the TLR4 pathway.
Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.
Mutation rates can evolve through genetic drift, indirect selection due to genetic hitchhiking, or direct selection on the physicochemical cost of high fidelity. However, for many systems, it has been difficult to disentangle the relative impact of these forces empirically. In RNA viruses, an observed correlation between mutation rate and virulence has led many to argue that their extremely high mutation rates are advantageous because they may allow for increased adaptability. This argument has profound implications because it suggests that pathogenesis in many viral infections depends on rare or de novo mutations. Here, we present data for an alternative model whereby RNA viruses evolve high mutation rates as a byproduct of selection for increased replicative speed. We find that a poliovirus antimutator, 3DG64S, has a significant replication defect and that wild-type (WT) and 3DG64S populations have similar adaptability in 2 distinct cellular environments. Experimental evolution of 3DG64S under selection for replicative speed led to reversion and compensation of the fidelity phenotype. Mice infected with 3DG64S exhibited delayed morbidity at doses well above the lethal level, consistent with attenuation by slower growth as opposed to reduced mutational supply. Furthermore, compensation of the 3DG64S growth defect restored virulence, while compensation of the fidelity phenotype did not. Our data are consistent with the kinetic proofreading model for biosynthetic reactions and suggest that speed is more important than accuracy. In contrast with what has been suggested for many RNA viruses, we find that within-host spread is associated with viral replicative speed and not standing genetic diversity.
N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.
We report an integrated workflow that allows mass spectrometry-based high-resolution hydroxyl radical protein footprinting (HR-HRPF) measurements to accurately measure the absolute average solvent accessible surface area (
CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-D-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-D-glucosamine-α-1,4-D-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-D-glucosamine-α-1,4-D-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract.
Oligo- and polysaccharides are infamous for being extremely flexible molecules, populating a series of well-defined rotational isomeric states under physiological conditions. Characterization of this heterogeneous conformational ensemble has been a major obstacle impeding high-resolution structure determination of carbohydrates and acting as a bottleneck in the effort to understand the relationship between the carbohydrate structure and function. This challenge has compelled the field to develop and apply theoretical and experimental methods that can explore conformational ensembles by both capturing and deconvoluting the structural and dynamic properties of carbohydrates. This review focuses on computational approaches that have been successfully used in combination with experiment to detail the three-dimensional structure of carbohydrates in a solution and in a complex with proteins. In addition, emerging experimental techniques for three-dimensional structural characterization of carbohydrate-protein complexes and future challenges in the field of structural glycobiology are discussed. The review is divided into five sections: (1) The complexity and plasticity of carbohydrates, (2) Predicting carbohydrate-protein interactions, (3) Calculating relative and absolute binding free energies for carbohydrate-protein complexes, (4) Emerging and evolving techniques for experimental characterization of carbohydrate-protein structures, and (5) Current challenges in structural glycoscience.
Glycoprotein gp120 is a surface antigen and virulence factor of human immunodeficiency virus 1. Broadly neutralizing antibodies (bNAbs) that react to gp120 from a variety of HIV isolates offer hope for the development of broadly effective immunogens for vaccination purposes, if the interactions between gp120 and bNAbs can be understood. From a structural perspective, gp120 is a particularly difficult system because of its size, the presence of multiple flexible regions, and the large amount of glycosylation, all of which are important in gp120-bNAb interactions. Here, the interaction of full-length, glycosylated gp120 with bNAb b12 is probed using high-resolution hydroxyl radical protein footprinting (HR-HRPF) by fast photochemical oxidation of proteins. HR-HRPF allows for the measurement of changes in the average solvent accessible surface area of multiple amino acids without the need for measures that might alter the protein conformation, such as mutagenesis. HR-HRPF of the gp120-b12 complex coupled with computational modeling shows a novel extensive interaction of the V1/V2 domain, probably with the light chain of b12. Our data also reveal HR-HRPF protection in the C3 domain caused by interaction of the N330 glycan with the b12 light chain. In addition to providing information about the interactions of full-length, glycosylated gp120 with b12, this work serves as a template for the structural interrogation of full-length glycosylated gp120 with other bNAbs to better characterize the interactions that drive the broad specificity of the bNAb.
Glycosylation patterns commonly change in cancer, resulting in expression of tumor-associated carbohydrate antigens (TACA). While promising, currently available anti-glycan antibodies are not useful for clinical cancer therapy. Here, we show that potent anti-glycan antibodies can be engineered to acquire cancer therapeutic efficacy. We designed yeast surface display to generate and select for therapeutic antibodies against the TACA SLea (CA19-9) in colon and pancreatic cancers. Elite clones showed increased affinity, better specificity, improved binding of human pancreatic and colon cancer cell lines, and increased complement-dependent therapeutic efficacy. Molecular modeling explained the structural basis for improved antibody functionality at the molecular level. These new tools of directed molecular evolution and selection for effective anti-glycan antibodies, provide insights into the mechanisms of cancer therapy targeting glycosylation, and provide major methodological advances that are likely to open up innovative avenues of research in the field of cancer theranostics.
Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.
Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE).
The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation. Taken together, these data can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.
Cell surface glycans are essential for establishing cell communication, adhesion, and migration. However, it remains challenging to obtain cell surface-specific information about glycoconjugate structures. Acquiring this information is essential for unraveling the functional role of glycans and for exploiting them as clinical targets. To specifically analyze the N-glycoprotein forms expressed at the cell surface, we developed a C18 liquid chromatography (LC)-mass spectrometry (MS)-based glycoproteomics method in combination with highly specific cell surface protein labeling and enrichment using a biotin label. The surface-specificity of the method was validated by MS-based proteomics of subcellular component marker proteins. Using the human keratinocytes N/TERT-1 as a model system, we identified and quantified the glycosylation of hundreds of cell surface N-glycosylation sites. This approach allowed us to study the glycoforms present at the functional relevant cell surface, omitting immaturely glycosylated proteins present in the secretory pathway. Interestingly, the different stages of N-glycan processing at individual sites displayed at the cell surface were found to correlate with their accessibility for ER-residing processing enzymes, as investigated through molecular dynamics simulations. Using the new approach, we compared N-glycosylation sites of proteins expressed on the cell surface to their counterparts in a total cell lysate, showing profound differences in glycosylation between the subcellular components and indicating the relevance of the method for future studies in understanding contextual glycan functions.
Select lectins have powerful anti-viral properties that effectively neutralize HIV-1 by targeting the dense glycan shield on the virus. Here, we reveal the mechanism by which one of the most potent lectins, BanLec, achieves its inhibition. We identify that BanLec recognizes a subset of high-mannose glycans via bidentate interactions spanning the two binding sites present on each BanLec monomer that were previously considered separate carbohydrate recognition domains. We show that both sites are required for high-affinity glycan binding and virus neutralization. Unexpectedly we find that BanLec adopts a tetrameric stoichiometry in solution whereby the glycan-binding sites are positioned to optimally target glycosylated viral spikes. The tetrameric architecture, together with bidentate binding to individual glycans, leads to layers of multivalency that drive viral neutralization through enhanced avidity effects. These structural insights will prove useful in engineering successful lectin therapeutics targeting the dense glycan shield of HIV.
Standard infectious disease practice calls for aggressive drug treatment that rapidly eliminates the pathogen population before resistance can emerge. When resistance is absent, this elimination strategy can lead to complete cure. However, when resistance is already present, removing drug-sensitive cells as quickly as possible removes competitive barriers that may slow the growth of resistant cells. In contrast to the elimination strategy, a containment strategy aims to maintain the maximum tolerable number of pathogens, exploiting competitive suppression to achieve chronic control. Here, we combine in vitro experiments in computer-controlled bioreactors with mathematical modeling to investigate whether containment strategies can delay failure of antibiotic treatment regimens. To do so, we measured the "escape time" required for drug-resistant Escherichia coli populations to eclipse a threshold density maintained by adaptive antibiotic dosing. Populations containing only resistant cells rapidly escape the threshold density, but we found that matched resistant populations that also contain the maximum possible number of sensitive cells could be contained for significantly longer. The increase in escape time occurs only when the threshold density-the acceptable bacterial burden-is sufficiently high, an effect that mathematical models attribute to increased competition. The findings provide decisive experimental confirmation that maintaining the maximum number of sensitive cells can be used to contain resistance when the size of the population is sufficiently large.
There is an urgent need for a vaccine with efficacy against SARS-CoV-2. We hypothesize that peptide vaccines containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation would drive both humoral and cellular immunity with high specificity, potentially avoiding undesired effects such as antibody-dependent enhancement (ADE). Additionally, such vaccines can be rapidly manufactured in a distributed manner. In this study, we combine computational prediction of T cell epitopes, recently published B cell epitope mapping studies, and epitope accessibility to select candidate peptide vaccines for SARS-CoV-2. We begin with an exploration of the space of possible T cell epitopes in SARS-CoV-2 with interrogation of predicted HLA-I and HLA-II ligands, overlap between predicted ligands, protein source, as well as concurrent human/murine coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, viral source protein abundance, sequence conservation, coverage of high frequency HLA alleles and co-localization of CD4+ and CD8+ T cell epitopes. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering to select regions with surface accessibility, high sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. From 58 initial candidates, three B cell epitope regions were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we propose a set of SARS-CoV-2 vaccine peptides for use in subsequent murine studies and clinical trials.
The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor's ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR's extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.
The processes by which pathogens evolve within a host dictate the efficacy of treatment strategies designed to slow antibiotic resistance evolution and influence population-wide resistance levels. The aim of this study is to describe the underlying genetic and phenotypic changes leading to antibiotic resistance within a patient who died as resistance evolved to available antibiotics. We assess whether robust patterns of collateral sensitivity and response to combinations existed that might have been leveraged to improve therapy.
Vancomycin-resistant Enterococcus (VRE) is a leading cause of hospital-acquired infections and continues to spread despite widespread implementation of pathogen-targeted control guidelines. Commensal gut microbiota provide colonization resistance to VRE, but the role of gut microbiota in VRE acquisition in at-risk patients is unknown. To address this gap in our understanding, we performed a case-control study of gut microbiota in hospitalized patients who did (cases) and did not (controls) acquire VRE. We matched case subjects to control subjects by known risk factors and "time at risk," defined as the time elapsed between admission until positive VRE screen. We characterized gut bacterial communities using 16S rRNA gene amplicon sequencing of rectal swab specimens. We analyzed 236 samples from 59 matched case-control pairs. At baseline, case and control subjects did not differ in gut microbiota when measured by community diversity (P = 0.33) or composition (P = 0.30). After hospitalization, gut communities of cases and controls differed only in the abundance of the Enterococcus-containing operational taxonomic unit (OTU), with the gut microbiota of case subjects having more of this OTU than time-matched control subjects (P = 0.01). Otherwise, case and control communities after the time at risk did not differ in diversity (P = 0.33) or community structure (P = 0.12). Among patients who became VRE colonized, those having the Blautia-containing OTU on admission had lower Enterococcus relative abundance once colonized (P = 0.004). Our results demonstrate that the 16S profile of the gut microbiome does not predict VRE acquisition in hospitalized patients, likely due to rapid and profound microbiota change. The gut microbiome does not predict VRE acquisition, but it may be associated with Enterococcus expansion, suggesting that these should be considered two distinct processes.IMPORTANCE The Centers for Disease Control and Prevention estimates that VRE causes an estimated 54,000 infections and 539 million dollars in attributable health care costs annually. Despite improvements in hand washing, environmental cleaning, and antibiotic use, VRE is still prevalent in many hospitals. There is a pressing need to better understand the processes by which patients acquire VRE. Multiple lines of evidence suggest that intestinal microbiota may help some patients resist VRE acquisition. In this large case-control study, we compared the 16S profile of intestinal microbiota on admission in patients that did and did not subsequently acquire VRE. The 16S profile did not predict subsequent VRE acquisition, in part due to rapid and dramatic change in the gut microbiome following hospitalization. However, Blautia spp. present on admission predicted decreased Enterococcus abundance after VRE acquisition, and Lactobacillus spp. present on admission predicted Enterococcus dominance after VRE acquisition. Thus, VRE acquisition and domination may be distinct processes.
Thrombospondin type-1 repeats (TSRs) are small protein motifs containing six conserved cysteines forming three disulfide bonds that can be modified with an O-linked fucose. Protein O-fucosyltransferase 2 (POFUT2) catalyzes the addition of O-fucose to TSRs containing the appropriate consensus sequence, and the O-fucose modification can be elongated to a Glucose-Fucose disaccharide with the addition of glucose by β3-glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in embryonic lethality in mice, highlighting the biological significance of O-fucose modification on TSRs. Knockout of POFUT2 in HEK293T cells has been shown to cause complete or partial loss of secretion of many proteins containing O-fucosylated TSRs. In addition, POFUT2 is localized to the endoplasmic reticulum (ER) and only modifies folded TSRs, stabilizing their structures. These observations suggest that POFUT2 is involved in an ER quality control mechanism for TSR folding and that B3GLCT also participates in quality control by providing additional stabilization to TSRs. However, the mechanisms by which addition of these sugars result in stabilization are poorly understood. Here, we conducted molecular dynamics (MD) simulations and provide crystallographic and NMR evidence that the Glucose-Fucose disaccharide interacts with specific amino acids in the TSR3 domain in thrombospondin-1 that are within proximity to the O-fucosylation modification site resulting in protection of a nearby disulfide bond. We also show that mutation of these amino acids reduces the stabilizing effect of the sugars in vitro. These data provide mechanistic details regarding the importance of O-fucosylation and how it participates in quality control mechanisms inside the ER.
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