A Zika virus-specific IgM elicited in pregnancy exhibits ultrapotent neutralization.

Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.

Acoustic force spectroscopy reveals subtle differences in cellulose unbinding behavior of carbohydrate-binding modules.

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein-carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often associated with microbial surface-tethered cellulosomes or secreted cellulase enzymes to enhance substrate accessibility. However, it is not well known how CBMs recognize, bind, and dissociate from polysaccharides to facilitate efficient cellulolytic activity, due to the lack of mechanistic understanding and a suitable toolkit to study CBM-substrate interactions. Our work outlines a general approach to study the unbinding behavior of CBMs from polysaccharide surfaces using a highly multiplexed single-molecule force spectroscopy assay. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and method for uniform deposition of insoluble polysaccharides on the AFS chip surfaces are demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surfaces suggests distinct multimodal CBM binding conformations, with structural mechanisms further explored using molecular dynamics simulations. Applying classical dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results also highlight critical limitations of applying classical theory to explain the highly multivalent binding interactions for cellulose-CBM bond rupture forces exceeding 15 pN.

Machine learning-driven multiscale modeling reveals lipid-dependent dynamics of RAS signaling proteins.

RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.

Zika Virus Replicates in the Vagina of Mice with Intact Interferon Signaling.

Zika virus (ZIKV) is unusual among flaviviruses in its ability to spread between humans through sexual contact, as well as by mosquitoes. Sexual transmission has the potential to change the epidemiology and geographic range of ZIKV compared to mosquito-borne transmission and potentially could produce distinct clinical manifestations, so it is important to understand the host mechanisms that control susceptibility to sexually transmitted ZIKV. ZIKV replicates poorly in wild-type mice following subcutaneous inoculation, so most ZIKV pathogenesis studies use mice lacking type I interferon (IFN-αß) signaling (e.g., Ifnar1-/-). We found that wild-type mice support ZIKV replication following intravaginal infection, consistent with prior studies, although the infection remained localized to the lower female reproductive tract. Vaginal ZIKV infection required a high-progesterone state (pregnancy or pretreatment with depot medroxyprogesterone acetate [DMPA]) even in Ifnar1-/- mice that otherwise are highly susceptible to ZIKV infection. Progesterone-mediated susceptibility did not appear to result from a compromised epithelial barrier, blunted antiviral gene induction, or changes in vaginal leukocyte populations, leaving open the mechanism by which progesterone confers susceptibility to vaginal ZIKV infection. DMPA treatment is a key component of mouse vaginal infection models for herpes simplex virus and Chlamydia, but the mechanisms by which DMPA increases susceptibility to those pathogens also remain poorly defined. Understanding how progesterone mediates susceptibility to ZIKV vaginal infection may provide insights into host mechanisms influencing susceptibility to diverse sexually transmitted pathogens. IMPORTANCE Zika virus (ZIKV) is transmitted by mosquitoes, similar to other flaviviruses. However, ZIKV is unusual among flaviviruses in its ability also to spread through sexual transmission. We found that ZIKV was able to replicate in the vaginas of wild-type mice, even though these mice do not support ZIKV replication by other routes, suggesting that the vagina is particularly susceptible to ZIKV infection. Vaginal susceptibility was dependent on a high-progesterone state, which is a common feature of mouse vaginal infection models for other pathogens, through mechanisms that have remained poorly defined. Understanding how progesterone mediates susceptibility to ZIKV vaginal infection may provide insights into host mechanisms that influence susceptibility to diverse sexually transmitted pathogens.

Visualization of the HIV-1 Env glycan shield across scales.

The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane.

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

Type 2 Diabetes Mellitus and Nonvalvular Atrial Fibrillation in Mexico: National Registries Raise a Red Flag.

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases worldwide and is highly prevalent in Mexico, as 10.2% of the adult population harbors this condition. T2DM is usually associated with cardiovascular comorbidities, including arrhythmias. Metabolic impairment is one of the mechanisms that contribute to tissue remodeling that affects atrial structure, and concomitant, the cardiac conduction system, both could result in atrial fibrillation (AF). AF is estimated to affect more than a half million Mexicans, and its incidence is expected to keep rising. According to national registries, T2DM is present in 28.4% of Mexican patients with AF and the coexistence of both diseases is associated with a higher risk of stroke. In clinical practice, the CHA2DS2-VASc risk score is useful for stroke risk stratification in patients with AF to facilitate the adequate use of anticoagulation therapy. T2DM is among the items of the CHA2DS2-VASc score because it correlates with an intrinsic prothrombotic state. In this narrative review, we present information that highlights the need for optimal glucose control and adequate anticoagulation in subjects with T2DM and AF.

Exploring CRD mobility during RAS/RAF engagement at the membrane.

During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.

Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III.

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM-cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1-cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.

The novel EhHSTF7 transcription factor displays an oligomer state and recognizes a heat shock element in the Entamoeba histolytica parasite.

The heat shock response is a conserved mechanism that allows cells to respond and survive stress damage and is transcriptionally regulated by the heat shock factors and heat shock elements. The P-glycoprotein confer the multidrug resistance phenotype; Entamoeba histolytica has the largest multidrug resistance gene family described so far; one of these genes, the EhPgp5 gene, has an emetine-inducible expression. A functional heat shock element was localized in the EhPgp5 gene promoter, indicating transcriptional regulation by heat shock factors. In this work, we determined the oligomer state of EhHSTF7 and the recognition of the heat shock element of the EhPgp5 gene. The EhHSTF7 recombinant protein was obtained as monomer and oligomer. In silico molecular docking predicts protein-DNA binding between EhHSTF7 and 5'-GAA-3' complementary bases. The rEhHSTF7 protein specifically binds to the heat shock element of the EhPgp5 gene in gel shift assays. The competition assays with heat shock element mutants indicate that 5'-GAA-3' complementary bases are necessary for the rEhHSTF7 binding. Finally, the siRNA-mediated knockdown of Ehhstf7 expression causes downregulation of EhPgp5 expression, suggesting that EhHSTF7 is likely to play a key role in the E. histolytica multidrug resistance. This is the first report of a transcription factor that recognizes a heat shock element from a gene involved in drug resistance in parasites. However, further analysis needs to demonstrate the biological relevance of the EhHSTF7 and the rest of the heat shock factors of E. histolytica, to understand the underlying regulation of transcriptional control in response to stress.

Development of Martini 2.2 parameters for N-glycans: a case study of the HIV-1 Env glycoprotein dynamics.

N-linked glycans are ubiquitous in nature and play key roles in biology. For example, glycosylation of pathogenic proteins is a common immune evasive mechanism, hampering the development of successful vaccines. Due to their chemical variability and complex dynamics, an accurate molecular understanding of glycans is still limited by the lack of effective resolution of current experimental approaches. Here, we have developed and implemented a reductive model based on the popular Martini 2.2 coarse-grained force field for the computational study of N-glycosylation. We used the HIV-1 Env as a direct applied example of a highly glycosylated protein. Our results indicate that the model not only reproduces many observables in very good agreement with a fully atomistic force field but also can be extended to study large amount of glycosylation variants, a fundamental property that can aid in the development of drugs and vaccines.

Unveiling the Dynamics of KRAS4b on Lipid Model Membranes.

Small GTPase proteins are ubiquitous and responsible for regulating several processes related to cell growth and differentiation. Mutations that stabilize their active state can lead to uncontrolled cell proliferation and cancer. Although these proteins are well characterized at the cellular scale, the molecular mechanisms governing their functions are still poorly understood. In addition, there is limited information about the regulatory function of the cell membrane which supports their activity. Thus, we have studied the dynamics and conformations of the farnesylated KRAS4b in various membrane model systems, ranging from binary fluid mixtures to heterogeneous raft mimics. Our approach combines long time-scale coarse-grained (CG) simulations and Markov state models to dissect the membrane-supported dynamics of KRAS4b. Our simulations reveal that protein dynamics is mainly modulated by the presence of anionic lipids and to some extent by the nucleotide state (activation) of the protein. In addition, our results suggest that both the farnesyl and the polybasic hypervariable region (HVR) are responsible for its preferential partitioning within the liquid-disordered (Ld) domains in membranes, potentially enhancing the formation of membrane-driven signaling platforms.

Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD.

CRAF activation requires binding to membrane-anchored and active GTP-bound RAS. Whereas its RAS-binding domain (RBD) contains the main binding interface to the RAS G domain, its cysteine-rich domain (CRD) is responsible for association to anionic lipid-rich membranes. Both RAF domains are connected by a short linker, and it remains unclear if the two domains act independently or if one domain can impact the function of the other. Here, we used a combination of coarse-grained and all-atom molecular dynamics simulations of a CRAF RBD-CRD construct to investigate the dynamics of the RBD when it is tethered to CRD that is anchored to a POPC:POPS model membrane. First, we show that the RBD positioning is very dynamic with a preferential localization near the membrane surface. Next, we show that membrane-localized RBD has its RAS-binding interface mostly inaccessible because of its proximity to the membrane. Several positively charged residues in this interface were identified from simulations as important for driving RBD association to the membrane. Surface plasmon resonance (SPR) measurements confirmed that mutations of these RBD residues reduced the liposome partitioning of RBD-CRD. Last, simulations indicated that the presence of RBD near the membrane led to a local enrichment of anionic lipids that could potentially enhance the membrane affinity of the entire RBD-CRD construct. This was supported by SPR measurements that showed stronger liposome partitioning of RBD-CRD relative to CRD alone. These findings thus suggest that the RBD and CRD have synergistic effects on their membrane dynamics, with CRD bringing RBD closer to the membrane that impacts its accessibility to RAS and with RBD causing local anionic lipid enrichment that enhances the overall affinity between the membrane and RBD-CRD. These mechanisms have potential implications on the order of events of the interactions between RAS and CRAF at the membrane.

Machine Learning Algorithm Identifies an Antibiotic Vocabulary for Permeating Gram-Negative Bacteria.

Drug discovery faces a crisis. The industry has used up the "obvious" space in which to find novel drugs for biomedical applications, and productivity is declining. One strategy to combat this is rational approaches to expand the search space without relying on chemical intuition, to avoid rediscovery of similar spaces. In this work, we present proof of concept of an approach to rationally identify a "chemical vocabulary" related to a specific drug activity of interest without employing known rules. We focus on the pressing concern of multidrug resistance in Pseudomonas aeruginosa by searching for submolecules that promote compound entry into this bacterium. By synergizing theory, computation, and experiment, we validate our approach, explain the molecular mechanism behind identified fragments promoting compound entry, and select candidate compounds from an external library that display good permeation ability.

Membrane perturbing properties of toxin mycolactone from Mycobacterium ulcerans.

Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin's distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin's interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin's preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin's cellular access, T-cell immunosuppression, and therapeutic potential.

Development of Envelope Protein Antigens To Serologically Differentiate Zika Virus Infection from Dengue Virus Infection.

Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (>12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients.

Lack of Durable Cross-Neutralizing Antibodies Against Zika Virus from Dengue Virus Infection.

Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence (>6 months after infection) is unknown. We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus-specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity.

Effect of Glycosylation on an Immunodominant Region in the V1V2 Variable Domain of the HIV-1 Envelope gp120 Protein.

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchange molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. These studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to focus the immune response on key neutralizing epitopes.

Membrane-mediated regulation of the intrinsically disordered CD3ϵ cytoplasmic tail of the TCR.

The regulation of T-cell-mediated immune responses depends on the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on T-cell receptors. Although many details of the signaling cascades are well understood, the initial mechanism and regulation of ITAM phosphorylation remains unknown. We used molecular dynamics simulations to study the influence of different compositions of lipid bilayers on the membrane association of the CD3ϵ cytoplasmic tails of the T-cell receptors. Our results show that binding of CD3ϵ to membranes is modulated by both the presence of negatively charged lipids and the lipid order of the membrane. Free-energy calculations reveal that the protein-membrane interaction is favored by the presence of nearby basic residues and the ITAM tyrosines. Phosphorylation minimizes membrane association, rendering the ITAM motif more accessible to binding partners. In systems mimicking biological membranes, the CD3ϵ chain localization is modulated by different facilitator lipids (e.g., gangliosides or phosphoinositols), revealing a plausible regulatory effect on activation through the regulation of lipid composition in cell membranes.

Residue Leu940 has a crucial role in the linkage and reaction specificity of the glucansucrase GTF180 of the probiotic bacterium Lactobacillus reuteri 180.

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu(940) in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu(940) of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67-100% of (α1→6) linkages) were produced by GTF180 and its Leu(940) mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu(940) in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.