Biology subjects

Granier, S.

Publications and source records attributed to Granier, S..

17 recordsLinked to original sources

bioRxiv2026

Distinct atypical chemokine receptor 1 determinants underlie bacterial toxins recognition and pore formation

Atypical chemokine receptor 1 (ACKR1) is one of the most promiscuous receptors in the human chemokine system, engaging structurally diverse chemokines through a conformationally flexible N-terminal tail. This same interface is exploited by pathogens, including Plasmodium vivax and Staphylococcus aureus (SA), via a compact sulfotyrosine code. Among pathogenic proteins recognizing ACKR1, the SA leukocidin pair HlgAB is a notable exception. HlgAB-mediated pore formation is only weakly competed by chemokines, the Duffy binding protein, or antibodies targeting the receptor's N-terminus, leaving open how HlgAB engage ACKR1. Combining structural biology approaches with cell-based assays, we show that HlgA and HlgB engage ACKR1 sulfated N-terminus with distinct affinities and site hierarchies, with a single higher-affinity site for sulfated tyrosine 41 present in HlgA but absent in HlgB. Unexpectedly, this N-terminal engagement is dispensable for pore formation; productive lysis instead requires a separate interaction between the toxins and ACKR1 extracellular vestibule. These results define a two-step recognition mechanism, toxin capture by the sulfotyrosine N-terminus followed by vestibule-dependent pore formation, and extend the view that ACKR1 promiscuity arises from distributed, ligand-specific use of multiple receptor surfaces rather than a single adaptable interface.

biochemistry↗

bioRxiv2026

Real-time analysis of pore formation by bi-component staphylococcal leukotoxins using the two-electrode voltage-clamp technique

Pore forming toxins (PFTs) are cytotoxins secreted in water-soluble form by pathogenic bacteria. They have the ability to form pores in the membrane of host cells, ultimately leading to cell death by lytic activity. Staphylococcus aureus produces a variety of bi-component PFTs, the leukocidins, which target and lyse particular leukocytes, erythrocytes and endothelial cells through specific interactions with membrane receptors. Most of these receptors belong to the family of complement or chemokine receptors that are G protein-coupled receptors (GPCRs). Gamma-hemolysins (Hlgs) are the major leukocidins secreted by S. aureus, and form receptor-dependent hetero-octameric pores through mechanisms that are not fully elucidated. Studying these molecular mechanisms is technically challenging due to the requirement of specific receptors in a lipid bilayer environment. In the present article, we developed a simple and highly sensitive method allowing cell surface expression of a large diversity of target receptors and recording in real-time, currents generated by neo-formed pores. This method is based on the heterologous expression of receptors in Xenopus oocytes and on the two-electrode voltage-clamp technique with electrophysiological robots. Using this approach, we characterized the concentration dependent-kinetics of pore formation, determined the receptor density as a limiting factor, showed specific response to non-cognate pairing of PFTs, observed cell surface binding of F subunits preceding pore formation and propose a hybrid model of subunit oligomerization. This method could be easily implemented for the in vitro characterization of various PFTs on a wide diversity of membrane receptors, to decipher early mechanisms of pore formation or to screen therapeutic agents blocking the cytotoxicity of receptor-dependent PFTs. Author SummaryStaphylococcus aureus is a bacterial species naturally present in our external flora and environment, but it is also one of the main pathogens responsible for nosocomial infections in hospital, with strains having highly problematic multi-resistance to antibiotics. S. aureus is able to secrete various virulence factors, some of which can specifically target and lyse our immune cells, making us more vulnerable to this pathogen. Thus, leukotoxins bind to receptors on the cell surface, drastically change their conformation and form cytotoxic pores in the membrane. Studying the molecular mechanisms underlying the formation of these pores is technically challenging due to their requirement for specific receptors. Here, we tested a simple electrophysiological method enabling the real-time measurement of pore formation on model cells (Xenopus oocytes), which express the receptors of interest. We were thus able to elucidate the kinetics of pore formation, the limiting role of receptors in this process, and propose a complementary model to the standard model. We also demonstrated the ability of this method to detect pore formation of non-cognate pairs of subunits and suggest further applications to characterize pore-forming properties of other toxins, to identify new target receptors, or to screen therapeutic agents inhibiting the formation of pores.

microbiology↗

bioRxiv2025

Deltaviruses spread through a viral Trojan Horse

Hepatitis D-like viral satellites, known as deltaviruses, have been recently discovered in a wide range of animals. These viruses are thought to expropriate glycoproteins from helper viruses to form infectious particles. Here, we challenge this paradigm and demonstrate that deltaviruses are packaged within helper virus particles, using them as viral Trojan Horses for cell entry. By leveraging orthogonal electronic and photonic super-resolution microscopy, we visualize deltaviruses enclosed within virions from rhabdo-, herpes-, and arenavirus families. We show that this conserved hitchhiking mechanism ensures concomitant deltavirus-helper virus spread, which advantages the dissemination of deltaviruses, broadens their host range and expands their tropism. Our findings reveal a previously unrecognized mode of viral transmission, providing a framework to investigate overlooked deltavirus infections outside of the human liver.

microbiology↗

bioRxiv2024

Inactive structures of the vasopressin V2 receptor reveal distinct antagonist binding modes for Tolvaptan and Mambaquaretin toxin

AbstractAntagonists of the arginine-vasopressin (AVP) V2 receptor (V2R) are key therapeutic compounds to treat hyponatremia or polycystic kidney diseases. Compounds such as Tolvaptan (TVP) and Conivaptan are marketed drugs but their mechanisms of inhibition are not known. In addition, TVP presents unwanted side effects such as serious liver injury. Conivaptan also targets AVP V1a receptor subtype and thus is not selective to V2R. To develop novel molecules with less side effects and better selectivity, rational drug design based on experimental three-dimensional G protein-coupled receptor (GPCR) structures is a powerful and successful avenue. The lack of antagonist-bound V2R structures has however impaired this strategy for this GPCR. To fill this gap of knowledge, we solved here the cryo-electron microscopy structures of the vasopressin V2R in complex with two selective antagonists, the nonpeptide TVP and the green mamba snake Mambaquaretin toxin (MQ1). Both ligands, known to be competitive, bind into the orthosteric AVP binding site but with substantial differences. The small molecule binds deeper than MQ1, and directly contacts the toggle switch residue W2846.48 in the transmembrane domain 6 (TM6), whereas the peptide Kunitz- fold toxin presents more extensive contacts with the receptor through additional interactions with extracellular and transmembrane residues. As anticipated from the pharmacological properties of TVP and MQ1, both structures represent inactive conformations of the V2R. Their comparison with those of the active AVP-bound V2R reveals the molecular mechanisms modulating receptor activity. Finally, the structure of the V2R bound to a mini-protein such as MQ1 opens a new pharmacology era in the field of water homeostasis and renal diseases.

pharmacology and toxicology↗

bioRxiv2024

Constitutive activity of an atypical chemokine receptor revealed by inverse agonistic nanobodies

Chemokine stimulation of atypical chemokine receptor 3 (ACKR3) does not activate G proteins but recruits arrestins. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that high constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive action of ACKR3 using novel inverse agonistic nanobodies to suppress basal activity. These new tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal, non-chemotactic, breast cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide a new therapeutic approach for targeting ACKR3.

biochemistry↗

bioRxiv2024

Physical interaction with Ephrin B1 promotes CXCR4 intracellular localization and oncogenic potential

Chemokine receptor 4 (CXCR4) is a member of the chemokine receptor exclusively activated by the chemokine CXCL12. While CXCR4 regulates numerous physiological processes associated with cell migration and embryogenesis, its overexpression has been involved in various cancer types. Studies suggest that intracellular CXCR4 expression rather than CXCR4-operated signaling underlies its pro-tumorigenic functions. Given the role of GPCR interacting proteins in their trafficking and subcellular localization, we characterized the CXCR4 interactome using an affinity purification coupled to mass spectrometry (AP-MS) strategy. The most abundant protein identified in the CXCR4 interactome is Ephrin B1, a member of the Ephrin protein family that shares several functions with CXCR4, such as the regulation of cell migration and proliferation. Further studies showed that interaction between CXCR4 and Ephrin B1 is direct and enhanced by treating cell with CXCL12. They also indicated that Ephrin B1 prevents CXCR4 N-glycosylation, decreases CXCR4 cell surface expression and consistently inhibits CXCL12-induced CXCR4 coupling to Gi1-3 and the recruitment of {beta}-arrestins 1 and 2. Conversely, Ephrin B1 signals to Erk1,2 through CXCR4 activation and mediates the decrease in Death Receptor 5 expression elicited by intracellular CXCR4. Collectively, these findings identify Ephrin B1 as a key mediator of CXCR4 tumorigenic signaling.

biochemistry↗

bioRxiv2023

Conformational dynamics underlying Atypical Chemokine Receptor 3 activation

Atypical Chemokine Receptor 3 (ACKR3) is a G protein-coupled receptor that does not signal through G proteins. It is known as a chemokine scavenger involved in various pathologies, making it an appealing yet intriguing therapeutic target. Indeed, the structural properties that govern ACKR3 functional selectivity and the overall conformational dynamics of ACKR3 activation are poorly understood. Here we combined Hydrogen/Deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics simulations to examine the binding mode and mechanism of action of various small-molecule ACKR3 ligands of different efficacy for {beta}-arrestin recruitment. Our results show that activation or inhibition of ACKR3 is largely governed by intracellular conformational changes of helix 6, intracellular loop 2 and helix 7, while the DRY motif becomes protected during both processes. Moreover, HDX-MS identifies the binding sites and the allosteric modulation of ACKR3 upon {beta}-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small-molecule ligands, the overall activation dynamics of ACKR3, the binding mode of {beta}-arrestin 1 and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

biochemistry↗

bioRxiv2023

Biased activation of the vasopressin V2 receptor probed by NMR, paramagnetic ligands, and molecular dynamics simulations

G protein-coupled receptors (GPCRs) control critical intercellular communications by responding to extracellular stimuli and undertaking conformational changes to convey signals to intracellular effectors. We combined NMR, molecular pharmacology, and molecular dynamics (MD) simulations to study the conformational diversity of the vasopressin V2 GPCR subtype (V2R) bound to different types of ligands: the antagonist tolvaptan, the endogenous unbiased agonist arginine-vasopressin, and MCF14, a Gs-protein biased agonist. We developed a double-labeling NMR scheme to study the conformational dynamics: V2R was subjected to lysine 13CH3 methylation, whereas the agonists were tagged with a paramagnetic probe. Paramagnetic relaxation enhancements were used to validate the ligand binding poses in the MD simulations. We found that the bias for the Gs protein over the {beta}-arrestin pathway involves interactions between the conserved NPxxY motif in the transmembrane helix (TM) 7 and a central hydrophobic patch in TM3, which constrains TM7 and likely inhibits {beta}-arrestin signaling. A similar mechanism was observed for the pathogenic mutation, I1303.43N, which constitutively activates the Gs protein without concomitant {beta}-arrestin recruitment. This mechanism resembles to opioid receptors findings indicating common patterns in class A GPCRs.

biophysics↗

bioRxiv2022

Structure of the vasopressin hormone-V2 receptor-β-arrestin1 ternary complex

Arrestins interact with G protein-coupled receptors (GPCRs) to stop G protein activation and to initiate key signaling pathways. Recent structural studies shed light on the molecular mechanisms involved in GPCR-arrestin coupling, but whether this process is conserved among GPCRs is poorly understood. Here, we report the cryo-electron microscopy active structure of the wild-type arginine-vasopressin V2 receptor (V2R) in complex with {beta}-arrestin1. It reveals an atypical position of {beta}-arrestin1 compared to previously described GPCR-arrestin assemblies, associated with an original V2R/{beta}-arrestin1 interface involving all receptor intracellular loops. Phosphorylated sites of the V2R C-terminus are clearly identified and interact extensively with the {beta}-arrestin1 N-lobe, in agreement with structural data obtained with chimeric or synthetic systems. Overall, these findings highlight a striking structural variability among GPCR-arrestin signaling complexes.

biochemistry↗

bioRxiv2021

Structural insights into recognition of chemokine receptors by Staphylococcus aureus leukotoxins

Staphylococcus aureus (SA) leukocidin LukED belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes and leukocytes. The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2 and CCR5 using a combination of structural, pharmacological and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining the available experimental information with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.

biochemistry↗

bioRxiv2021

Discovery and mechanism of action of small molecule inhibitors of ceramidases

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes such as energy utilisation and cell proliferation. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to ultimately produce pro-proliferative sphingosine-1-phosphate. Human ceramidases can be soluble proteins (acid and neutral ceramidase) or integral membrane proteins (alkaline ceramidases). Increasing ceramide levels to increase apoptosis has shown efficacy as a cancer treatment using small molecules inhibiting a soluble ceramidase. Due to the transmembrane nature of alkaline ceramidases, no specific small molecule inhibitors have been reported. Here, we report novel fluorescent substrates (FRETceramides) of ceramidases that can be used to monitor enzyme activity in real-time. We use FRETceramides to discover the first drug-like inhibitors of alkaline ceramidase 3 (ACER3) which are active in cell-based assays. Biophysical characterization of enzyme:inhibitor interactions reveal a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules. Table of contents summaryUse of synthetic fluorescent ceramide molecules allows the discovery of the first selective drug-like small molecule inhibitors for alkaline ceramidase 3, an intra-membrane enzyme involved in sphingolipid metabolism in health and disease.

biochemistry↗

bioRxiv2021

An automated platform for structural analysis of membrane proteins through serial crystallography

Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze at a structural level. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins due to lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperature and in a single support. We apply this approach to two human integral membrane proteins, which allowed us to capture different conformational states of intramembrane enzyme-product complexes and analyze the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting and serial crystallography enabling screening of small molecule libraries with membrane protein crystals grown in meso. This approach brings badly needed automation for this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. HighlightsO_LIA fully automated, online workflow enables rapid determination of membrane protein structures by serial X-ray crystallography (SSX). C_LIO_LIHigh resolution room temperature and cryogenic structures of ADIPOR2 provide insights into the dynamic nature of receptor:ligand interactions. C_LIO_LIA web-based application allows remote user-guided experimental design and execution. C_LIO_LIAn automated SSX-based ligand discovery pipeline for integral membrane proteins is introduced. C_LI

biochemistry↗

bioRxiv2021

Molecular insights into the μ-opioid receptorselective signaling

GPCR functional selectivity has opened new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the {beta}-arrestin pathways through the -opioid receptor (OR). We combined functional assays in living cells, solution NMR spectroscopy and enhanced-sampling molecular dynamic simulations to identify the specific OR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists triggered OR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair {beta}-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.

bioinformatics↗

bioRxiv2021

Mechanisms of GPCR hijacking by Staphylococcus aureus

Atypical chemokine receptor 1 (ACKR1) is a G protein-coupled receptor (GPCR) targeted by Staphylococcus aureus (SA) bi-component pore-forming leukotoxins to promote bacterial growth and immune evasion. Here we have developed an integrative molecular pharmacology and structural biology approach in order to characterize the effect of leukotoxins HlgA and HlgB on ACKR1 structure and function. Interestingly, we found that both components HlgA and HlgB compete with endogenous chemokines through a direct binding to ACKR1 captured by native mass spectrometry (MS). Unexpectedly, HDX-MS analysis revealed that toxin binding allosterically modulates the intracellular G protein-binding domain of the receptor, resulting in dissociation of ACKR1-G protein complexes in living cells. Altogether, our study brings important molecular insights into the initial steps of leukotoxins targeting a host GPCR. Our findings may open the way to develop antibiotics inhibiting host receptors binding, a mechanism of action less prone to resistance.

biochemistry↗

bioRxiv2020

Structure of the antidiuretic hormone vasopressin receptor signalling complex.

Arginine-vasopressin (AVP) is a neurohypophysial peptide known as the antidiuretic hormone. It forms an active signalling complex with the V2 receptor (V2R) and the Gs protein, promoting a cAMP/PKA-dependent aquaporin insertion in apical membranes of principal cells of the renal collecting ducts and ultimately, water reabsorption. Molecular mechanisms underlying activation of this critical G protein-coupled receptor (GPCR) signalling system are still unknown. To fill this gap of knowledge, we report here the structure of the AVP-V2R-Gs complex using cryo-electron microscopy (cryo-EM). Single-particle analysis revealed the presence of three different states. The two best maps were combined with computational and NMR spectroscopy constraints to reconstruct two structures of the ternary complex. These structures differ in AVP and Gs binding modes and could thus represent distinct complex conformations along the signalling activation pathway. Importantly, as compared to those of other class A GPCR-Gs complexes, the structures revealed an original receptor-Gs interface in which the Gs subunit penetrates deeper into the active V2R, notably forming an ionic bond between its free C-terminal carboxylic function and the side chain of R137 in the V2R. Interestingly, the structures help to explain how V2R R137H or R137L/C variants can lead to two severe genetic diseases with opposite clinical outcomes, cNDI or NSIAD respectively. Our study thus provides important structural insights into the function of this clinically relevant GPCR signalling complex.

biochemistry↗

bioRxiv2019

Global phylogenomics of multidrug resistant Salmonella enterica serotype Kentucky ST198

Salmonella enterica serotype Kentucky (S. Kentucky) can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S. Kentucky ST198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug resistant (MDR) S. Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island 1 (variant SGI1-K) conferring resistance ampicillin, streptomycin, gentamicin, sulfamethoxazole, and tetracycline. Phylogeographic analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone resistance determining regions (QRDR) of DNA gyrase (gyrA) and DNA topoisomerase (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone's evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems, and/or azithromycin. These results indicate that most MDR S. Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials.

microbiology↗

bioRxiv2018

Structure of a human intramembrane ceramidase explains enzymatic dysfunction found in leukodystrophy

Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.

biochemistry↗