Insights into the Interaction Landscape of the EVH1 Domain of Mena.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed ß7-ß8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 µM). Inhibitor 6c displayed tighter binding (Kd ∼ 0.3 µM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

A hemoprotein with a zinc-mirror heme site ties heme availability to carbon metabolism in cyanobacteria.

Heme has a critical role in the chemical framework of the cell as an essential protein cofactor and signaling molecule that controls diverse processes and molecular interactions. Using a phylogenomics-based approach and complementary structural techniques, we identify a family of dimeric hemoproteins comprising a domain of unknown function DUF2470. The heme iron is axially coordinated by two zinc-bound histidine residues, forming a distinct two-fold symmetric zinc-histidine-iron-histidine-zinc site. Together with structure-guided in vitro and in vivo experiments, we further demonstrate the existence of a functional link between heme binding by Dri1 (Domain related to iron 1, formerly ssr1698) and post-translational regulation of succinate dehydrogenase in the cyanobacterium Synechocystis, suggesting an iron-dependent regulatory link between photosynthesis and respiration. Given the ubiquity of proteins containing homologous domains and connections to heme metabolism across eukaryotes and prokaryotes, we propose that DRI (Domain Related to Iron; formerly DUF2470) functions at the molecular level as a heme-dependent regulatory domain.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Discovery of a Biotin Synthase That Utilizes an Auxiliary 4Fe-5S Cluster for Sulfur Insertion.

Biotin synthase (BioB) is a member of the Radical SAM superfamily of enzymes that catalyzes the terminal step of biotin (vitamin B7) biosynthesis, in which it inserts a sulfur atom in desthiobiotin to form a thiolane ring. How BioB accomplishes this difficult reaction has been the subject of much controversy, mainly around the source of the sulfur atom. However, it is now widely accepted that the sulfur atom inserted to form biotin stems from the sacrifice of the auxiliary 2Fe-2S cluster of BioB. Here, we bioinformatically explore the diversity of BioBs available in sequence databases and find an unexpected variation in the coordination of the auxiliary iron-sulfur cluster. After in vitro characterization, including the determination of biotin formation and representative crystal structures, we report a new type of BioB utilized by virtually all obligate anaerobic organisms. Instead of a 2Fe-2S cluster, this novel type of BioB utilizes an auxiliary 4Fe-5S cluster. Interestingly, this auxiliary 4Fe-5S cluster contains a ligated sulfide that we propose is used for biotin formation. We have termed this novel type of BioB, Type II BioB, with the E. coli 2Fe-2S cluster sacrificial BioB representing Type I. This surprisingly ubiquitous Type II BioB has implications for our understanding of the function and evolution of Fe-S clusters in enzyme catalysis, highlighting the difference in strategies between the anaerobic and aerobic world.

Computational simulations of bispecific T cell engagers by a multiscale model.

The use of bispecific antibodies as T cell engagers can bypass the normal T cell receptor-major histocompatibility class interaction, redirect the cytotoxic activity of T cells, and lead to highly efficient tumor cell killing. However, this immunotherapy also causes significant on-target off-tumor toxicologic effects, especially when it is used to treat solid tumors. To avoid these adverse events, it is necessary to understand the fundamental mechanisms involved in the physical process of T cell engagement. We developed a multiscale computational framework to reach this goal. The framework combines simulations on the intercellular and multicellular levels. On the intercellular level, we simulated the spatial-temporal dynamics of three-body interactions among bispecific antibodies, CD3 and tumor-associated antigens (TAAs). The derived number of intercellular bonds formed between CD3 and TAAs was further transferred to the multicellular simulations as the input parameter of adhesive density between cells. Through the simulations under various molecular and cellular conditions, we were able to gain new insights into how to adopt the most appropriate strategy to maximize the drug efficacy and avoid the off-target effect. For instance, we discovered that the low antibody-binding affinity resulted in the formation of large clusters at the cell-cell interface, which could be important to control the downstream signaling pathways. We also tested different molecular architectures of the bispecific antibody and suggested the existence of an optimal length in regulating the T cell engagement. Overall, the current multiscale simulations serve as a proof-of-concept study to help in the future design of new biological therapeutics.

Immune activation of the p75 neurotrophin receptor: implications in neuroinflammation.

Despite structural similarity with other tumor necrosis factor receptor superfamily (TNFRSF) members, the p75 neurotrophin receptor (p75NTR, TNFR16) mediates pleiotropic biological functions not shared with other TNFRs. The high level of p75NTR expression in the nervous system instead of immune cells, its utilization of co-receptors, and its interaction with soluble dimeric, rather than soluble or cell-tethered trimeric ligands are all characteristics which distinguish it from most other TNFRs. Here, we compare these attributes to other members of the TNFR superfamily. In addition, we describe the recent evolutionary adaptation in B7-1 (CD80), an immunoglobulin (Ig) superfamily member, which allows engagement to neuronally-expressed p75NTR. B7-1-mediated binding to p75NTR occurs in humans and other primates, but not lower mammals due to specific sequence changes that evolved recently in primate B7-1. This discovery highlights an additional mechanism by which p75NTR can respond to inflammatory cues and trigger synaptic elimination in the brain through engagement of B7-1, which was considered to be immune-restricted. These observations suggest p75NTR does share commonality with other immune co-modulatory TNFR family members, by responding to immunoregulatory cues. The evolution of primate B7-1 to bind and elicit p75NTR-mediated effects on neuronal morphology and function are discussed in relationship to immune-driven modulation of synaptic actions during injury or inflammation.

Phosphate Binding in PNP Alters Transition-State Analogue Affinity and Subunit Cooperativity.

Purine nucleoside phosphorylases (PNPs) catalyze the phosphorolysis of 6-oxypurine nucleosides with an HPO42- dianion nucleophile. Nucleosides and phosphate occupy distinct pockets in the PNP active site. Evaluation of the HPO42- site by mutagenesis, cooperative binding studies, and thermodynamic and structural analysis demonstrate that alterations in the HPO42- binding site can render PNP inactive and significantly impact subunit cooperativity and binding to transition-state analogue inhibitors. Cooperative interactions between the cationic transition-state analogue and the anionic HPO42- nucleophile demonstrate the importance of reforming the transition-state ensemble for optimal inhibition with transition-state analogues. Altered phosphate binding in the catalytic site mutants helps to explain one of the known lethal PNP deficiency syndromes in humans.

Chemoenzymatic Synthesis of 3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP).

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin during the early stages of the innate immune response. ddhCTP has been shown to act as a chain terminator of flavivirus RNA-dependent RNA polymerases. To date, synthesis of ddhCTP requires complicated synthetic protocols or isolation of the enzyme viperin to catalyze the production of ddhCTP from CTP. Recombinant viperin approaches preclude the production of highly pure ddhCTP (free of contaminants such as CTP), whereas the chemical synthesis involves techniques or equipment not readily available to most laboratories. Herein, we describe the chemoenzymatic synthesis of ddhCTP, starting from commercially available ddhC. We utilize these methods to produce milligram quantities of ddhCTP, ddhCDP, and ddhCMP. Using purified semisynthetic ddhCTP and fully synthetic ddhCTP, we also show ddhCTP does not inhibit NAD+-dependent enzymes such as glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, or lactate dehydrogenase, contrary to a recent report.

Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection.

Andes virus (ANDV) and Sin Nombre virus (SNV) are the etiologic agents of severe hantavirus cardiopulmonary syndrome (HCPS) in the Americas for which no FDA-approved countermeasures are available. Protocadherin-1 (PCDH1), a cadherin-superfamily protein recently identified as a critical host factor for ANDV and SNV, represents a new antiviral target; however, its precise role remains to be elucidated. Here, we use computational and experimental approaches to delineate the binding surface of the hantavirus glycoprotein complex on PCDH1's first extracellular cadherin repeat domain. Strikingly, a single amino acid residue in this PCDH1 surface influences the host species-specificity of SNV glycoprotein-PCDH1 interaction and cell entry. Mutation of this and a neighboring residue substantially protects Syrian hamsters from pulmonary disease and death caused by ANDV. We conclude that PCDH1 is a bona fide entry receptor for ANDV and SNV whose direct interaction with hantavirus glycoproteins could be targeted to develop new interventions against HCPS.

Understanding the General Principles of T Cell Engagement by Multiscale Computational Simulations.

The use of bispecific antibodies as T cell engagers can bypass the normal TCR-MHC interaction, redirect the cytotoxic activity of T-cells, and lead to highly efficient tumor cell killing. However, this immunotherapy also causes significant on-target off-tumor toxicologic effects, especially when they were used to treat solid tumors. In order to avoid these adverse events, it is necessary to understand the fundamental mechanisms during the physical process of T cell engagement. We developed a multiscale computational framework to reach this goal. The framework combines simulations on the intercellular and multicellular levels. On the intercellular level, we simulated the spatial-temporal dynamics of three-body interactions among bispecific antibodies, CD3 and TAA. The derived number of intercellular bonds formed between CD3 and TAA were further transferred into the multicellular simulations as the input parameter of adhesive density between cells. Through the simulations under various molecular and cellular conditions, we were able to gain new insights of how to adopt the most appropriate strategy to maximize the drug efficacy and avoid the off-target effect. For instance, we discovered that the low antibody binding affinity resulted in the formation of large clusters at the cell-cell interface, which could be important to control the downstream signaling pathways. We also tested different molecular architectures of the bispecific antibody and suggested the existence of an optimal length in regulating the T cell engagement. Overall, the current multiscale simulations serve as a prove-of-concept study to help the future design of new biological therapeutics. SIGNIFICANCE: T-cell engagers are a class of anti-cancer drugs that can directly kill tumor cells by bringing T cells next to them. However, current treatments using T-cell engagers can cause serious side-effects. In order to reduce these effects, it is necessary to understand how T cells and tumor cells interact together through the connection of T-cell engagers. Unfortunately, this process is not well studied due to the limitations in current experimental techniques. We developed computational models on two different scales to simulate the physical process of T cell engagement. Our simulation results provide new insights into the general properties of T cell engagers. The new simulation methods can therefore serve as a useful tool to design novel antibodies for cancer immunotherapy.

SARS-CoV-2 multi-antigen protein microarray for detailed characterization of antibody responses in COVID-19 patients.

Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target multiple epitopes on different domains of the spike protein, and other SARS-CoV-2 proteins. We developed a SARS-CoV-2 multi-antigen protein microarray with the nucleocapsid, spike and its domains (S1, S2), and variants with single (D614G, E484K, N501Y) or double substitutions (N501Y/Deletion69/70), allowing a more detailed high-throughput analysis of the antibody repertoire following infection. The assay was demonstrated to be reliable and comparable to ELISA. We analyzed antibodies from 18 COVID-19 patients and 12 recovered convalescent donors. The S IgG level was higher than N IgG in most of the COVID-19 patients, and the receptor-binding domain of S1 showed high reactivity, but no antibodies were detected against the heptad repeat domain 2 of S2. Furthermore, antibodies were detected against S variants with single and double substitutions in COVID-19 patients who were infected with SARS-CoV-2 early in the pandemic. Here we demonstrated that the SARS-CoV-2 multi-antigen protein microarray is a powerful tool for detailed characterization of antibody responses, with potential utility in understanding the disease progress and assessing current vaccines and therapies against evolving SARS-CoV-2.

A non-neutralizing glycoprotein B monoclonal antibody protects against herpes simplex virus disease in mice.

There is an unmet need for monoclonal antibodies (mAbs) for prevention or as adjunctive treatment of herpes simplex virus (HSV) disease. Most vaccine and mAb efforts focus on neutralizing antibodies, but for HSV this strategy has proven ineffective. Preclinical studies with a candidate HSV vaccine strain, ΔgD-2, demonstrated that non-neutralizing antibodies that activate Fcγ receptors (FcγRs) to mediate antibody-dependent cellular cytotoxicity (ADCC) provide active and passive protection against HSV-1 and HSV-2. We hypothesized that this vaccine provides a tool to identify and characterize protective mAbs. We isolated HSV-specific mAbs from germinal center and memory B cells and bone marrow plasmacytes of ΔgD-2-vaccinated mice and evaluated these mAbs for binding, neutralizing, and FcγR-activating activity and for protective efficacy in mice. The most potent protective mAb, BMPC-23, was not neutralizing but activated murine FcγRIV, a biomarker of ADCC. The cryo-electron microscopic structure of the Fab-glycoprotein B (gB) assembly identified domain IV of gB as the epitope. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcγRIII when engineered as a human IgG1. These results highlight the importance of FcR-activating antibodies in protecting against HSV.

Cell-impermeable staurosporine analog targets extracellular kinases to inhibit HSV and SARS-CoV-2.

Herpes simplex virus (HSV) receptor engagement activates phospholipid scramblase triggering Akt translocation to the outer leaflet of the plasma membrane where its subsequent phosphorylation promotes viral entry. We hypothesize that this previously unrecognized outside-inside signaling pathway is employed by other viruses and that cell-impermeable kinase inhibitors could provide novel antivirals. We synthesized a cell-impermeable analog of staurosporine, CIMSS, which inhibited outer membrane HSV-induced Akt phosphorylation and blocked viral entry without inducing apoptosis. CIMSS also blocked the phosphorylation of 3-phosphoinositide dependent protein kinase 1 and phospholipase C gamma, which were both detected at the outer leaflet following HSV exposure. Moreover, vesicular stomatitis virus pseudotyped with SARS-CoV-2 spike protein (VSV-S), but not native VSV or VSV pseudotyped with Ebola virus glycoprotein, triggered this scramblase-Akt outer membrane signaling pathway. VSV-S and native SARS-CoV-2 infection were inhibited by CIMSS. Thus, CIMSS uncovered unique extracellular kinase processes linked to HSV and SARS-CoV-2 entry.

Human immunomodulatory ligand B7-1 mediates synaptic remodeling via the p75 neurotrophin receptor.

Cell surface receptors, ligands, and adhesion molecules underlie development, circuit formation, and synaptic function of the central nervous system and represent important therapeutic targets for many neuropathologies. The functional contributions of interactions between cell surface proteins of neurons and nonneuronal cells have not been fully addressed. Using an unbiased protein-protein interaction screen, we showed that the human immunomodulatory ligand B7-1 (hB7-1) interacts with the p75 neurotrophin receptor (p75NTR) and that the B7-1:p75NTR interaction is a recent evolutionary adaptation present in humans and other primates, but absent in mice, rats, and other lower mammals. The surface of hB7-1 that engages p75NTR overlaps with the hB7-1 surface involved in CTLA-4/CD28 recognition, and these molecules directly compete for binding to p75NTR. Soluble or membrane-bound hB7-1 altered dendritic morphology of cultured hippocampal neurons, with loss of the postsynaptic protein PSD95 in a p75NTR-dependent manner. Abatacept, an FDA-approved therapeutic (CTLA-4-hFc fusion) inhibited these processes. In vivo injection of hB7-1 into the murine subiculum, a hippocampal region affected in Alzheimer's disease, resulted in p75NTR-dependent pruning of dendritic spines. Here, we report the biochemical interaction between B7-1 and p75NTR, describe biological effects on neuronal morphology, and identify a therapeutic opportunity for treatment of neuroinflammatory diseases.

A glycan-based approach to cell characterization and isolation: Hematopoiesis as a paradigm.

Cell surfaces display a wide array of molecules that confer identity. While flow cytometry and cluster of differentiation (CD) markers have revolutionized cell characterization and purification, functionally heterogeneous cellular subtypes remain unresolvable by the CD marker system alone. Using hematopoietic lineages as a paradigm, we leverage the extraordinary molecular diversity of heparan sulfate (HS) glycans to establish cellular "glycotypes" by utilizing a panel of anti-HS single-chain variable fragment antibodies (scFvs). Prospective sorting with anti-HS scFvs identifies functionally distinct glycotypes within heterogeneous pools of mouse and human hematopoietic progenitor cells and enables further stratification of immunophenotypically pure megakaryocyte-erythrocyte progenitors. This stratification correlates with expression of a heptad of HS-related genes that is reflective of the HS epitope recognized by specific anti-HS scFvs. While we show that HS glycotyping provides an orthogonal set of tools for resolution of hematopoietic lineages, we anticipate broad utility of this approach in defining and isolating novel, viable cell types across diverse tissues and species.

HSMotifDiscover: identification of motifs in sequences composed of non-single-letter elements.

SUMMARY: The functional sub-string(s) of a biopolymer sequence defines the specificity of its interaction with other biomolecules and is often referred to as motifs. Computational algorithms and software have been broadly developed for finding such motifs in sequences in which the individual elements are single characters, such as those in DNA and protein sequences. However, there are more complex scenarios where the motifs exist in non-single-letter contexts, e.g. preferred patterns of chemical modifications on proteins, DNAs, RNAs or polysaccharides. To search for those motifs, we describe a new method that converts the modified sequence elements to representative single-letter codes and then uses a modified Gibbs-sampling algorithm to define the position specific scoring matrix representing the motif(s). As a proof of principle, we describe the implementation and application of an R package for discovering heparan sulfate (HS) motifs in glycan sequences, which are important in regulating protein-protein interactions. This software can be valuable for analyzing high-throughput glycoprotein binding data using microarrays with HS oligosaccharides or other biological polymers. AVAILABILITY AND IMPLEMENTATION: HSMotifDiscover is freely available as an open source R package released under an MIT license at https://github.com/bioinfoDZ/HSMotifDiscover and also available in the form of an app at https://hsmotifdiscover.shinyapps.io/HSMotifDiscover_ShinyApp/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.