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.

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.

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.

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.

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.

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.

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.

Structural and Functional Characterization of a Biliverdin-Binding Near-Infrared Fluorescent Protein From the Serpin Superfamily.

Biliverdin-binding serpins (BBSs) are proteins that are responsible for coloration in amphibians and fluoresce in the near-infrared (NIR) spectral region. Here we produced the first functional recombinant BBS of the polka-dot treefrog Boana punctata (BpBBS), assembled with its biliverdin (BV) chromophore, and report its biochemical and photochemical characterization. We determined the crystal structure of BpBBS at 2.05 Å resolution, which demonstrated its structural homology to the mammalian protease inhibitor alpha-1-antitrypsin. BV interaction with BpBBS was studied and it was found that the N-terminal polypeptide (residues 19-50) plays a critical role in the BV binding. By comparing BpBBS with the available NIR fluorescent proteins and expressing it in mammalian cells, we demonstrated its potential as a NIR imaging probe. These results provide insight into the non-inhibitory function of serpins, provide a basis for improving their performance in mammalian cells, and suggest possible paths for the development of BBS-based fluorescent probes.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

T cell receptor-targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice.

To delineate the in vivo role of different costimulatory signals in activating and expanding highly functional virus-specific cytotoxic CD8+ T cells, we designed synTacs, infusible biologics that recapitulate antigen-specific T cell activation signals delivered by antigen-presenting cells. We constructed synTacs consisting of dimeric Fc-domain scaffolds linking CD28- or 4-1BB-specific ligands to HLA-A2 MHC molecules covalently tethered to HIV- or CMV-derived peptides. Treatment of HIV-infected donor PBMCs with synTacs bearing HIV- or CMV-derived peptides induced vigorous and selective ex vivo expansion of highly functional HIV- and/or CMV-specific CD8+ T cells, respectively, with potent antiviral activities. Intravenous injection of HIV- or CMV-specific synTacs into immunodeficient mice intrasplenically engrafted with donor PBMCs markedly and selectively expanded HIV-specific (32-fold) or CMV-specific (46-fold) human CD8+ T cells populating their spleens. Notably, these expanded HIV- or CMV-specific CD8+ T cells directed potent in vivo suppression of HIV or CMV infections in the humanized mice, providing strong rationale for consideration of synTac-based approaches as a therapeutic strategy to cure HIV and treat CMV and other viral infections. The synTac platform flexibility supports facile delineation of in vivo effects of different costimulatory signals on patient-derived virus-specific CD8+ T cells, enabling optimization of individualized therapies, including HIV cure strategies.

Peptide-HLA-based immunotherapeutics platforms for direct modulation of antigen-specific T cells.

Targeted pharmacologic activation of antigen-specific (AgS) T cells may bypass limitations inherent in current T cell-based cancer therapies. We describe two immunotherapeutics platforms for selective delivery of costimulatory ligands and peptide-HLA (pHLA) to AgS T cells. We engineered and deployed on these platforms an affinity-attenuated variant of interleukin-2, which selectively expands oligoclonal and polyfunctional AgS T cells in vitro and synergizes with CD80 signals for superior proliferation versus peptide stimulation.

Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB.

Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.

Structural Insight into the Substrate Scope of Viperin and Viperin-like Enzymes from Three Domains of Life.

Viperin is a member of the radical S-adenosylmethionine superfamily and has been shown to restrict the replication of a wide range of RNA and DNA viruses. We recently demonstrated that human viperin (HsVip) catalyzes the conversion of CTP to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP or ddh-synthase), which acts as a chain terminator for virally encoded RNA-dependent RNA polymerases from several flaviviruses. Viperin homologues also exist in non-chordate eukaryotes (e.g., Cnidaria and Mollusca), numerous fungi, and members of the archaeal and eubacterial domains. Recently, it was reported that non-chordate and non-eukaryotic viperin-like homologues are also ddh-synthases and generate a diverse range of ddhNTPs, including the newly discovered ddhUTP and ddhGTP. Herein, we expand on the catalytic mechanism of mammalian, fungal, bacterial, and archaeal viperin-like enzymes with a combination of X-ray crystallography and enzymology. We demonstrate that, like mammalian viperins, these recently discovered viperin-like enzymes operate through the same mechanism and can be classified as ddh-synthases. Furthermore, we define the unique chemical and physical determinants supporting ddh-synthase activity and nucleotide selectivity, including the crystallographic characterization of a fungal viperin-like enzyme that utilizes UTP as a substrate and a cnidaria viperin-like enzyme that utilizes CTP as a substrate. Together, these results support the evolutionary conservation of the ddh-synthase activity and its broad phylogenetic role in innate antiviral immunity.

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

Immune responses to SARS-CoV-2 infection in hospitalized pediatric and adult patients.

Children and youth infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have milder disease than do adults, and even among those with the recently described multisystem inflammatory syndrome, mortality is rare. The reasons for the differences in clinical manifestations are unknown but suggest that age-dependent factors may modulate the antiviral immune response. We compared cytokine, humoral, and cellular immune responses in pediatric (children and youth, age <24 years) (n = 65) and adult (n = 60) patients with coronavirus disease 2019 (COVID-19) at a metropolitan hospital system in New York City. The pediatric patients had a shorter length of stay, decreased requirement for mechanical ventilation, and lower mortality compared to adults. The serum concentrations of interleukin-17A (IL-17A) and interferon-γ (IFN-γ), but not tumor necrosis factor-α (TNF-α) or IL-6, were inversely related to age. Adults mounted a more robust T cell response to the viral spike protein compared to pediatric patients as evidenced by increased expression of CD25+ on CD4+ T cells and the frequency of IFN-γ+ CD4+ T cells. Moreover, serum neutralizing antibody titers and antibody-dependent cellular phagocytosis were higher in adults compared to pediatric patients with COVID-19. The neutralizing antibody titer correlated positively with age and negatively with IL-17A and IFN-γ serum concentrations. There were no differences in anti-spike protein antibody titers to other human coronaviruses. Together, these findings demonstrate that the poor outcome in hospitalized adults with COVID-19 compared to children may not be attributable to a failure to generate adaptive immune responses.

In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

Redesigning HVEM Interface for Selective Binding to LIGHT, BTLA, and CD160.

Herpes virus entry mediator (HVEM) regulates positive and negative signals for T cell activation through co-signaling pathways. Dysfunction of the HVEM co-signaling network is associated with multiple pathologies related to autoimmunity, infectious disease, and cancer, making the associated molecules biologically and therapeutically attractive targets. HVEM interacts with three ligands from two different superfamilies using two different binding interfaces. The engagement with ligands CD160 and B- and T-lymphocyte attenuator (BTLA), members of immunoglobulin superfamily, is associated with inhibitory signals, whereas inflammatory responses are regulated through the interaction with LIGHT from the TNF superfamily. We computationally redesigned the HVEM recognition interfaces using a residue-specific pharmacophore approach, ProtLID, to achieve switchable-binding specificity. In subsequent cell-based binding assays the new interfaces, designed with only single or double mutations, exhibited selective binding to only one or two out of the three cognate ligands.

Narrow-Spectrum Antibiotic Targeting of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis.

Antibiotic resistance continues to spread at an alarming rate, outpacing the introduction of new therapeutics and threatening to globally undermine health care. There is a crucial need for new strategies that selectively target specific pathogens while leaving the majority of the microbiome untouched, thus averting the debilitating and sometimes fatal occurrences of opportunistic infections. To address these challenges, we have adopted a unique strategy that focuses on oxygen-sensitive proteins, an untapped set of therapeutic targets. MqnE is a member of the radical S-adenosyl-l-methionine (RS) superfamily, all of which rely on an oxygen-sensitive [4Fe-4S] cluster for catalytic activity. MqnE catalyzes the conversion of didehydrochorismate to aminofutalosine in the essential menaquinone biosynthetic pathway present in a limited set of species, including the gut pathogen Helicobacter pylori (Hp), making it an attractive target for narrow-spectrum antibiotic development. Indeed, we show that MqnE is inhibited by the mechanism-derived 2-fluoro analogue of didehydrochorismate (2F-DHC) due to accumulation of a radical intermediate under turnover conditions. Structures of MqnE in the apo and product-bound states afford insight into its catalytic mechanism, and electron paramagnetic resonance approaches provide direct spectroscopic evidence consistent with the predicted structure of the radical intermediate. In addition, we demonstrate the essentiality of the menaquinone biosynthetic pathway and unambiguously validate 2F-DHC as a selective inhibitor of Hp growth that exclusively targets MqnE. These data provide the foundation for designing effective Hp therapies and demonstrate proof of principle that radical SAM proteins can be effectively leveraged as therapeutic targets.