Biology subjects

Nagarajan, V.

Publications and source records attributed to Nagarajan, V..

16 recordsLinked to original sources

bioRxiv2025

Gut microbial interaction networks control autoimmunity to neuroretina

The gut microbiome influences the development of immune-mediated inflammatory diseases, including autoimmune uveitis, a sight-threatening ocular inflammation driven by retina-specific T cells1. Using a model of spontaneous autoimmune uveitis (sEAU) we showed that gut commensals provide immune stimuli that trigger disease2. Here we report that uveitis-promoting microbes are present in human gut flora and that colonization of germ-free (GF) mice with commensals from healthy human donors was sufficient to provoke disease. Severity of sEAU correlated with expansion of Akkermansia and contraction of short-chain fatty acid (SCFA)-producing Firmicutes, followed by decreased SCFA levels and a dominant gut Th1 effector response. Mechanistic gain-of-function experiments, enriching GF sEAU mice with Akkermansia, reproduced these microbiome, metabolite and immune phenotype shifts, and exacerbated disease, suggesting that Akkermansia promotes autoimmunity by outcompeting SCFA-producers and enhancing Th1-type responses. An inverse correlation between Akkermansia (Verrucomicrobia) and Firmicutes was also present in patients with uveitis, multiple sclerosis and Crohns disease. These findings reveal a stereotypic gut microbial interaction network that regulates systemic immune balance, and may represent an ecologically conserved mechanism through which the gut microbiome modulates autoimmune and inflammatory diseases.

immunology↗

bioRxiv2025

Time-Resolved Crystallography Reveals the Mechanisms of GTP hydrolysis for N-RAS and the Oncogenic Mutants G12C, G12V and Q61L

The RAS family of small GTPases are molecular switches that convey downstream signals regulating cell proliferation, differentiation, and apoptosis. The signaling competent GTP-bound RAS transitions to its inactive GDP-bound form through {gamma}-phosphate hydrolysis. Oncogenic RAS mutations hamper GTP hydrolysis and are present in up to 30% of all human cancers. Structural studies of RAS proteins bound to non-hydrolysable GTP analogs have revealed snapshots of the enzyme in its possibly active form. Yet, the mechanism of GTP hydrolysis has not been structurally resolved. To visualize this reaction in real time, we performed time-resolved crystallographic experiments employing a photolabile caged-GTP substrate. Fifty-seven distinctive reaction intermediates were captured during hydrolysis of a live GTP for N-RAS, the oncogenic mutants G12C, G12V and Q61L; and Y32R, a fast hydrolytic mutant. The reaction mechanisms and rates for the native and each of the mutants differed significantly; however, they shared common elements: an initially catalytically-defective open state, which transitions into the closed Michaelis complex state with solvent-assisted O3B-P{gamma} bond lengthening and breaking, followed by the release of the Mg2+ stabilized PO3-/PO4-3 species and unfolding of the switch loops. Given the conserved nature of GTP- and ATP-ases active sites, this structural work lays the basis to understand the universal mechanism of {gamma}-phosphate hydrolysis. Furthermore, search for cryptic binding sites during GTP hydrolysis in G12C, G12V, and Q61L mutants reveals the presence of distinctive state-dependent binding pockets that could be targets for structure-based drug discovery of experimentally resolved intermediates states.

cancer biology↗

bioRxiv2025

A C. elegans model of familial Alzheimer's disease shows age-dependent synaptic degeneration independent of amyloid β-peptide

The membrane-embedded {gamma}-secretase complex is involved in the intramembrane cleavage of [~] 150 substrates. Cleavage of amyloid precursor protein (APP)-derived substrate C99 generates 38-43-residue secreted amyloid {beta}-peptides (A{beta}), with the aggregation-prone 42-residue form (A{beta}42) particularly implicated in the pathogenesis of Alzheimers Disease (AD). However, whether A{beta}42 is the primary driver of neurodegeneration in AD remains unclear. Dominant mutations in APP or presenilin--the catalytic component of {gamma}-secretase--cause early-onset familial AD (FAD) and reduce one or more steps in the multi-step processive proteolysis of C99 to A{beta} peptides, apparently through stabilization of {gamma}-secretase enzyme-substrate (E-S) complexes. To investigate mechanisms of neurodegeneration in FAD, we developed new C. elegans models co-expressing wild-type or FAD-mutant C99 substrate and presenilin-1 (PSEN1) variants in neurons, allowing intramembrane processing of C99 to A{beta} in vivo. We demonstrate that while FAD-mutation of either C99 or PSEN1 leads to age-dependent synaptic loss, proteolytically inactive PSEN1 did not. Designed mutations that allow stable E-S complex formation without A{beta}42 or A{beta} production likewise result in synaptic degeneration. Moreover, replacement of C99 with variants of a Notch1-based substrate revealed that disrupted processing of another {gamma}-secretase substrate can similarly lead to synaptic degeneration. These results support a model in which synaptic loss can be triggered by toxic, stalled {gamma}-secretase E-S complexes in the absence of A{beta} production and not by simple loss of proteolytic function. This new C. elegans system provides a powerful platform to study the role of dysfunctional {gamma}-secretase substrate processing in FAD pathogenesis. SIGNIFICANCEDominantly inherited familial Alzheimers disease (FAD) is caused by mutations in the enzyme or substrate that produces amyloid {beta}-peptides (A{beta}). These mutations alter enzyme processing of substrate to A{beta} and can skew production of A{beta} to aggregation-prone forms that deposit as pathological plaques. Nevertheless, whether A{beta} is the primary driver of AD remains unresolved. Recent evidence supports a model in which loss of neuronal connections in FAD is due to the stalled enzymatic process of A{beta} production, rather than the A{beta} products. Using the roundworm C. elegans as a genetic model system, we show here that the stalled process itself, rather than A{beta} or reduced enzyme activity, can trigger loss of neuronal connections with age in this simple model animal.

neuroscience↗

bioRxiv2025

Identification of Novel Marine-based Inhibitors against Tetracycline Destructase in Acinetobacter Baumannii using Computational Approaches

Tetracyclines are indispensable antibiotics employed to treat a broad spectrum of bacterial infections. However, the emergence of clinical pathogens exhibiting significant resistance to these drugs has posed a formidable challenge in managing bacterial diseases effectively. This resistance is attributed to the proliferation and diversity of resistance genes and various mechanisms that render tetracyclines inactive. A prevalent mechanism of resistance is enzymatic inactivation by tetracycline destructases, which compromises the efficacy of tetracyclines. Consequently, there is a pressing need to discover novel molecules capable of inhibiting tetracycline destructases activity and thereby enhancing the drugs potency. In this study, we identify novel inhibitors for tetracycline destructase (TDase) in Acinetobacter Baumannii, utilizing compounds derived from marine environments. Through a high-throughput virtual screening approach, we have investigated the potent natural marine compounds interaction and dynamic behaviour of tetracycline destructase through molecular simulations. These findings hold promise for the development of novel and efficacious therapeutics against bacterial infections.

bioinformatics↗

bioRxiv2025

Chromosome-scale genome assembly of B10.RIII, an autoimmune susceptible mouse strain

BackgroundA vast majority of potential drug candidates are initially tested in mice before human trials, resulting in life-saving treatments and preventive measures. Hundreds of autoimmune disorders, including arthritis, lupus etc., affect millions of people all over the world. Studying those disorders using mouse models is crucial for understanding the disease mechanisms. Knowing the genome sequence of these mouse models would help with those vital studies. B10.RIII is one of the most susceptible mouse strains used as a model to understand autoimmune diseases but did not have its genome sequenced until now. FindingsIn this study, we generated the first, high-quality chromosome level genome sequence of B10.RIII, using a combination of long read, short read and optical mapping techniques. The B10.RIII genome sequence scored higher than the reference mouse genome both in assembly completeness and assembly quality. The B10.RIII genome annotation based on the reference genome identified about 98% of the known reference genes. ConclusionsWe believe the availability of B10.RIII genome sequence will help advance the understanding of autoimmune diseases, giving the researchers a better idea on its unique genetic makeup. Comparative studies of B10.RIII with other mouse models could also significantly enhance the power of these models towards their clinical applications.

immunology↗

bioRxiv2025

SCassist: An AI Based Workflow Assistant for Single-Cell Analysis

SummarySingle-cell RNA sequencing (scRNA-seq) data analysis often involves complex iterative workflow, requiring significant expertise and time. To navigate this complexity, we have developed SCassist, an R package that leverages the power of the large language models (LLMs) to guide and enhance scRNA-seq analysis. SCassist integrates LLMs into key workflow steps, to analyze user data and provide relevant recommendations for filtering, normalization and clustering parameters. It also provides LLM guided insightful interpretations of variable features and principal components, along with cell type annotations and enrichment analysis. SCassist provides intelligent assistance using popular LLMs like Googles Gemini, OpenAIs GPT and Metas Llama3, making scRNA-seq analysis accessible to researchers at all levels. Availability and implementationThe SCassist package, along with the detailed tutorials, is available at GitHub. https://github.com/NIH-NEI/SCassist

bioinformatics↗

bioRxiv2025

Commensal-derived Trehalose Monocorynomycolate Triggers γδ T Cell-driven Protective Ocular Barrier Immunity

Commensals shape host physiology through molecular crosstalk with host receptors. Identifying specific microbial factors that causally influence host immunity is key to understanding homeostasis at the host-microbe interface and advancing microbial-based therapeutics. Here, we identify trehalose monocorynomycolate (TMCM) from Corynebacterium mastitidis (C. mast) as a potent stimulator of IL-17 production by {gamma}{delta} T cells at the ocular surface. Mechanistically, TMCM-driven IL-17 responses require both IL-1 signals and {gamma}{delta} TCR signaling, which also supports endogenous {gamma}{delta} T cell IL-1R1 expression. Notably, synthetic TMCM alone is sufficient to mimic the effect of C. mast in inducing {gamma}{delta} T cell immunity and protect against pathogenic corneal infection. Our findings establish TMCM as a key mediator of commensal-driven immune defense, highlighting its potential as a {gamma}{delta} T cell adjuvant and a microbiome-informed therapeutic to enhance IL-17-driven protection at barrier sites such as the ocular surface. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/643820v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@63139forg.highwire.dtl.DTLVardef@9d6978org.highwire.dtl.DTLVardef@775e37org.highwire.dtl.DTLVardef@ca84b6_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LICorynomycolates enable ocular C. mast colonization and protective IL-17 immunity C_LIO_LITMCM drives IL-17 from {gamma}{delta} T cells through TCR and IL-1R signaling C_LIO_LI{gamma}{delta} TCR signaling maintains the expression of endogenous IL-1R1 C_LIO_LISynthetic TMCM mimics the ability of C. mast to induce {gamma}{delta} T cell immunity in the eye C_LIO_LITMCM protects against P. aeruginosa keratitis, highlighting its therapeutic potential C_LI

immunology↗

bioRxiv2025

IAN: An Intelligent System for Omics Data Analysis and Discovery

IAN is an R package that addresses the challenge of integrating, analyzing and interpreting high-throughput "omics" data, using a multi-agent artificial intelligence (AI) system. IAN leverages popular pathway and regulatory datasets (KEGG, WikiPathways, Reactome, GO, ChEA) and the STRING database for protein-protein interactions to perform standard enrichment analysis. The individual enrichment results are then used to generate insightful summaries, for each of the datasets, using a large language model (LLM) through a multi-agent architecture. These summaries are then contextually integrated and interpreted by the LLM, guided by carefully engineered prompts and grounding instructions, to provide insightful explanations, system overview, key regulators, novel observations etc. We demonstrate IANs potential to facilitate biological discovery from complex omics data, by reanalyzing two already published data and evaluating the results. We also show remarkable performance of IAN, in terms of avoiding hallucination. IAN package, along with installation instructions and example usage, is available on https://github.com/NIH-NEI/IAN.

bioinformatics↗

bioRxiv2025

AI-Driven Analysis Unveils Functional Dynamics of Müller Cells in Autoimmune Inflammation

Muller cells are the most abundant glial cell type in the human and mouse retina, playing a crucial role in maintaining retinal homeostasis. However, many aspects of Muller cell function remain poorly characterized. In this study, we reanalyzed a single-cell RNA-seq (scRNA-seq) dataset from Aire-/- mice, focusing on Muller cells and T cells. We identified nine distinct Muller cell subgroups and five T cell subgroups, with activated Muller cells comprising the majority of the Muller cells in the inflamed retina. Using SCassist, an AI-based workflow assistant for single-cell analysis, we created a comparison matrix to quantify pathway involvement in each Muller cell subset. This approach unveils the functional dynamics of Muller cells during retinal inflammation. Activated Muller cells primarily exhibit enhanced inflammatory activity and adopt a macrophage or dendritic-cell-like phenotype, in the presence or absence of increased neuronal activity. These changes are primarily driven by Interferon Regulatory Factors (IRFs), acting alone or in concert with Neuronal Differentiation 1 (NEUROD1). We further inferred the interactions between Muller cells and T cells and found that activated Muller cells do not appear to exhibit extra chemoattraction to Th1 cells compared to other Muller cell subsets, but they do show such effects on the Th1-like regulatory T cells (Tregs). Activated Muller cells display nearly exclusive expression of immune checkpoint molecules, primarily targeting Th1 cells. These findings may uncover a previously unrecognized role for activated Muller cells in attenuating Th1 cell activity.

immunology↗

bioRxiv2025

Ocular immune privilege in action: the living eye imposes unique regulatory and anergic gene signatures on uveitogenic T cells

Despite ocular immune privilege, circulating retina-specific T cells can trigger autoimmune uveitis, yet intraocular bleeding--a relatively common event--rarely leads to disease. Using an in vivo immune privilege model, we previously reported that all naive retina-specific T cells entering the eye become primed in situ; about Ob% become FoxpO+ T-regulatory cells (Tregs), while the rest fail to induce pathology. Here, single-cell transcriptomics and functional validation revealed distinct phenotypes in both populations: ocular Tregs were highly suppressive, whereas non-Tregs expressed suppression- and anergy-associated genes and lacked regulatory function. Trajectory analyses suggested that Tregs and anergic cells arise from a common proliferative precursor in parallel, rather than sequentially. Our data indicate a key checkpoint governing the divergence of anergic and regulatory fates. These findings provide molecular-level insights into ocular immune privilege and may inform strategies to silence autoimmune effector cells or reverse T cell unresponsiveness in cancer, vaccination, or chronic infection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/640701v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1e13f5org.highwire.dtl.DTLVardef@b065feorg.highwire.dtl.DTLVardef@f83014org.highwire.dtl.DTLVardef@1ca8f0_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIEye-primed retina-specific T cells develop distinct tolerance-associated phenotypes. C_LIO_LIEye-induced anergic T cells remain hyporesponsive to antigen re-stimulation. C_LIO_LIRegulatory and anergic T cells differentiate in parallel from a common precursor. C_LIO_LIInduction of T cell anergy is a novel feature of ocular immune privilege. C_LI

immunology↗

bioRxiv2025

Transcriptomic Profiling of Old Age Sarcoma Patients using TCGA RNA-seq data

Sarcoma is a rare malignancy with poor prognosis, especially in older patients ([≥] 65 years) as seen in our preliminary analysis and some previous studies. Moreover, these patients have limited treatment options due to therapy-associated adverse effects and altered tumor micro-environment, which could be associated with their lower prognosis. Studying the underlying biology that drives cancer progression in these patients will help design personalized therapy and improve outcomes for them. This study aims to analyze TCGA-SARC RNA-seq data for characterizing the transcriptomic profile of older age (OA: [≥] 65 years) compared to younger age (YA: 18-65 years) sarcoma patients. RNA-seq and clinical data of sarcoma patients were acquired from TCGA, and the samples were grouped as OA ([≥] 65 years) and YA (18-65 years) patients. Differential gene expression analysis, pathway analysis, transcription factor enrichment analysis, gene-specific survival analysis and network analysis were performed. When comparing the gene expression profiles of the 108 OA and 154 YA patients, significant differentially regulated genes (n=733), transcription factors (n=10), hub genes (n=10) and the pathways that characterize the former were identified. Furthermore, 16 dysregulated genes were found that were significantly associated with a poor prognosis in OA sarcoma patients. In accordance with existing evidence of an altered tumor microenvironment in older-age cancer patients, the identified significant genes are associated with the regulation of certain important tumorigenic pathways such as EMT (epithelial-to-mesenchymal transition), calcium signaling, angiogenesis, ECM (extracellular matrix) degradation, Wnt/{beta}-catenin pathways, suggesting the potential cause for lower prognosis in the OA patients. Thus, these findings pave the way to characterize the OA sarcoma patients which can be validated by multi-omics analysis and clinical studies in the future, in turn providing improved treatment options and survival for the same.

cancer biology↗

bioRxiv2024

TLR2 Supports γδ T cell IL-17A Response to ocular surface commensals by Metabolic Reprogramming

The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival {gamma}{delta} T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival V{gamma}4 T cells through a combination of {gamma}{delta} TCR ligation and IL-1 signaling. Here, we identify V{gamma}6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that V{gamma}6 cells require not only extrinsic (via dendritic cells) but also intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes responsible for metabolic shift to fatty acid oxidation to support Il17a transcription. We identify one key transcription factor, I{kappa}B{zeta}, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal {gamma}{delta} T cells. SummaryOcular commensal Corynebacterium mastitidis (C. mast) induces IL-17 responses from {gamma}{delta} T cells by activating TLR2 signaling. {gamma}{delta} T cell-intrinsic TLR2 stimulation can increase Il17a transcription and promote fatty acid oxidation, favoring {gamma}{delta} T cell IL-17A responses. Highlights(1) V{gamma}6 T cells are a major C. mast-responsive subset in the conjunctiva (2) TLR2-deficient mice exhibit reduced {gamma}{delta} T cell responses to ocular commensal bacteria. (3) {gamma}{delta} T cell-intrinsic TLR2 deficiency causes defects of fatty acid oxidation and IL-17A production in a {gamma}{delta} subset-specific manner. (4) The transcription factor, I{kappa}B{zeta} is upregulated by TLR2 stimulation and supports {gamma}{delta} IL-17A production through fatty acid oxidation. O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/587519v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1d1ba87org.highwire.dtl.DTLVardef@2d17d9org.highwire.dtl.DTLVardef@78e846org.highwire.dtl.DTLVardef@1d96dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Alzheimers disease is characterized pathologically by cerebral deposition of 42-residue amyloid {beta}-peptide (A{beta}42), proteolytically produced from amyloid precursor protein (APP) by {beta}- and {gamma}-secretases.1 Although mutations in APP and presenilin, the catalytic component of {gamma}-secretase, cause familial Alzheimers disease (FAD), a role for A{beta}42 as the primary disease driver has not been clearly established and remains controversial.2,3 Here we show through comprehensive analysis of the multi-step proteolysis of APP substrate C99 by {gamma}-secretase that FAD mutations are consistently deficient in early proteolytic events, not later events that produce secreted A{beta} peptides. Cryo-electron microscopy revealed that a substrate mimetic traps {gamma}-secretase at the transition state for intramembrane proteolysis, and this structure closely aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and fluorescence lifetime imaging microscopy in cultured cells support stabilization by FAD mutations of enzyme-substrate and/or enzyme-intermediate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorabditis elegans led to age-dependent synaptic loss only when one of the transgenes carried an FAD mutation. Designed mutations that stabilize the enzyme-substrate complex and block proteolysis likewise led to synaptic loss. Collectively, these findings implicate the stalled process--not the released products--of {gamma}-secretase cleavage of substrates in FAD pathogenesis.

neuroscience↗

bioRxiv2023

Single-cell multiomic analysis reveals the involvement of Type I interferon-responsive CD8+ T cells in amyloid beta-associated memory loss

Alzheimers disease (AD) is the leading cause of dementia worldwide, but there are limited therapeutic options and no current cure. While the involvement of microglia in AD has been highly appreciated, the role of other innate and adaptive immune cells remains largely unknown, partly due to their scarcity and heterogeneity. This study aimed to study non-microglial immune cells in wild type and AD-transgenic mouse brains across different ages. Our results uncovered the presence of a unique CD8+ T cell population that were selectively increased in aging AD mouse brains, here referred to as "disease-associated T cells (DATs)". These DATs were found to express an elevated tissue-resident memory and Type I interferon-responsive gene signature. Further analysis of aged AD mouse brains showed that these CD8+ T cells were not present in peripheral or meningeal tissues. Preventing CD8+ T cell development in AD-transgenic mice via genetic deletion of beta-2 microglobulin (B2m) led to a reduction of amyloid-{beta} plaque formation in aged mice, and improved memory in AD-transgenic mice as early as four months of age. The integration of transcriptomic and epigenomic profiles at the single-cell level revealed potential transcription factors that reshape the regulomes of CD8+ T cells. These findings highlight a critical role for DATs in the progression of AD and provide a new avenue for treatment.

immunology↗

bioRxiv2023

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling

Initiation of B-cell receptor (BCR)1 signaling, and subsequent antigen-encounter in germinal centers2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL)4 and lymphoma5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells6-9, the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKC{delta} and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKC{delta}10-12, SHIP113,14 and SHP114, 15,16, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKC{delta}-mediated phosphorylation of CD25 on its cytoplasmic tail (S268). Genetic rescue experiments identified CD25-S268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

immunology↗

bioRxiv2018

Molecular mimicry in deoxy-nucleotide catalysis: the structure of Escherichia coli dGTPase reveals the molecular basis of dGTP selectivity

Deoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly-located on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 [A] cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 [A]) of single stranded DNA in an allosteric site primes the active site by conformationally \"opening\" a tyrosine gate allowing enhanced substrate binding.\n\nSignificance StatementdNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 -which blocks reverse transcription of HIV-1 in macrophages- Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 [A] cleft separates SAMHD1 residues from dNTP.

biophysics↗