Biology subjects

England, P.

Publications and source records attributed to England, P..

11 recordsLinked to original sources

bioRxiv2026

Structural insights into the inactive state of the adhesion GPCR ADGRV1

Adhesion G protein-coupled receptors (aGPCRs) are involved in numerous physiological processes, including cell-cell and cell-matrix interactions, and are associated with several human diseases. ADGRV1 is a member of the aGPCR family and plays a significant role in the sensorineural systems. Mutations of ADGRV1 are linked to the Usher syndrome, a genetic disorder causing deafness and blindness in human. However, the molecular mechanisms that control the activity of ADGRV1 remain unclear. In this study, we present the high-resolution cryo-electron microscopy structure of the inactive ADGRV1 receptor in complex with the nanobody RE02, providing detailed insights into its transmembrane domain, intracellular loop conformations and the inactive orthosteric site. Functional cellular assays revealed that ADGRV1 exhibits a weak constitutive activation independent of the tethered agonist peptide, primarily coupling with Gi proteins. These findings suggest that ADGRV1 may employ an alternative activation mechanism, distinct that from other aGPCRs reported so far. Our structural analysis highlights that ADGRV1 does not follow the conventional tethered agonist activation mechanism. This is due to the divergent sequence of its tethered agonist peptide, as well as the lack of residues critical for conformational transitions toward the active state in other aGPCRs. Moreover, the large intracellular loop 3 (ICL3) adopts a closed conformation, tightly packed against the transmembrane region of ADGRV1. This suggests that the ICL3 loop can compete with G-protein binding, ultimately acting as an additional barrier for ADGRV1 activation. Significance StatementAdhesion G protein-coupled receptors (aGPCRs) regulate essential processes such as cell communication and sensory function, yet the mechanisms controlling many family members remain poorly understood. We report the first high-resolution cryo-electron microscopy structure of the human receptor ADGRV1, a protein linked to Usher syndrome, a major genetic cause of deafness and blindness. Structural and functional analyses reveal that ADGRV1 displays weak constitutive signaling and likely operates through an activation mechanism distinct from the canonical tethered agonist model that defines most aGPCRs. Unique structural features, including a closed intracellular loop (ICL3) that may limit G-protein engagement, suggest an alternative regulatory strategy. These findings expand the conceptual framework of aGPCR activation and provide a foundation for understanding ADGRV1 function in sensory physiology and disease.

biochemistry↗

bioRxiv2024

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.

molecular biology↗

bioRxiv2023

A monoclonal antibody collection for C. difficile typing

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in adults. Various C. difficile strains circulate currently, associated with different outcomes and antibiotic resistance profiles. However, most studies still focus on the reference strain 630 that does not circulate anymore, partly due to the lack of immunological tools to study current clinically important C. difficile PCR ribotypes. Herein, we immunized mice expressing human variable antibody genes with the Low Molecular Weight (LMW) subunit of the surface layer protein SlpA from various C. difficile strains. Monoclonal antibodies purified from hybridomas bound LMW with high-affinity and whole bacteria from current C. difficile ribotypes with different cross-specificities. This first collection of anti-C. difficile mAbs represent valuable tools for basic and clinical research.

immunology↗

bioRxiv2023

Anti-S-layer monoclonal antibodies impact Clostridioides difficile physiology

Clostridioides difficile (C. difficile), a gram-positive anaerobic and spore-forming bacterium, is the leading cause of nosocomial antibiotic-associated diarrhea in adults and is characterized by high levels of recurrence and mortality. Surface-layer Protein A (SlpA), the most expressed protein on bacterial surface, plays a crucial role in the early stages of infection although its role in C. difficile physiology is yet to be fully understood. Anti-S-layer antibodies have been identified in the sera of convalescent patients and correlate with improved outcome of C. difficile infection (CDI). However, the precise mechanisms of how anti-S-layer antibodies can confer protection to the host remain unknown. In this study, we report the first monoclonal antibodies (mAbs) targeting S-layer of the reference strain 630. Characterization of these mAbs unravels important roles for S-layer protein in growth, toxin secretion, and biofilm formation with, surprisingly, opposite effects of different anti-SlpA mAbs on these functions. One anti-SlpA mAb impaired C. difficile growth and restored sensitivity to lysozyme-induced lysis. These findings suggest that anti-S-layer antibody responses may include protective and detrimental effects for the host and provide important insights for designing adequate S-layer-targeting therapeutics.

microbiology↗

bioRxiv2023

Raynals, an online tool for the analysis of dynamic light scattering

Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size distribution profile of samples containing microscopic particles in suspension or solubilised polymers. In this work, we introduce Raynals, an user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov-Phillips regularisation. Performance is evaluated on simulated and experimental data, generated by different DLS instruments, for several proteins and gold nanoparticles. DLS data can be easily misinterpreted and the simulation tools available in Raynals allow understanding of the limitations of the measurement and its resolution. It has been designed as a tool to address quality control of biological samples, during sample preparation and optimisation, and it helps in the detection of aggregates showing the influence of large particles. Last, Raynals provides flexibility in the way the data is presented, allows exporting publication-quality figures, it is free for academic use, and can be accessed online on the eSPC data analysis platform at spc.embl-hamburg.de.

biophysics↗

bioRxiv2023

An original potentiating mechanism revealed by the cryoEM structures of the human α7 nicotinic receptor in complex with nanobodies

The human 7 nicotinic receptor is a pentameric channel mediating cellular and neuronal communication. It has attracted considerable interest to design ligands for the treatment of neurological and psychiatric disorders. To develop a novel class of 7 ligands, we recently generated two nanobodies named E3 and C4 acting as positive and silent allosteric modulators respectively. Here, we solved the cryo-EM structures of the nanobody-receptor complexes. E3 and C4 bind to a common epitope involving two subunits at the apex of the receptor. They form by themselves a symmetric pentameric assembly that extends the extracellular domain. Unlike C4, the binding of E3 drives an active or desensitized conformation in the absence of orthosteric agonist, and mutational analysis shows a key contribution of a N-linked sugar moiety in mediating E3 potentiation. The nanobody E3, by remotely controlling the global allosteric conformation of the receptor, implements an original mechanism of regulation which opens new avenues for drug design.

neuroscience↗

bioRxiv2022

Molecular mechanisms regulating the pH-dependent pr/E interaction in yellow fever virus.

Flavivirus particles bud in the ER of infected cells as immature virions composed of 180 heterodimers of glycoproteins prM and E, associated as 60 (prM/E)3 trimeric spikes. Exposure to the mildly acidic pH of the TGN results in dissociation of the trimeric spikes followed by reassociation of the prM/E protomers into 90 dimers organized in a characteristic herringbone pattern. The furin site in prM is exposed in the dimers for maturation of prM into M and pr. For flaviviruses such as the tick-borne encephalitis virus (TBEV) as well as for dengue virus, it was shown that at neutral pH pr loses affinity for E, such that it dissociates from the mature particle as soon as it reaches the external milieu, which is at neutral pH. Using a soluble recombinant form of E (sE) and pr from yellow fever virus (YFV), we show here that the affinity of pr for recombinant E protein remains high even at neutral pH. The X-ray structure of YFV pr/sE shows more extensive inter-chain hydrogen bonding than does the dengue or TBEV, and also that it retains the charge complementarity between the interacting surfaces of the two proteins even at neutral pH. We further show that pr blocks sE flotation with liposomes when exposed at low pH at a 1:1 stoichiometry, yet in the context of the virus particle, an excess of 10:1 pr:E ratio is required to block virus/liposome fusion. In aggregate, our results show that the paradigm obtained from earlier studies of other flaviviruses does not apply to yellow fever virus, the flavivirus type species. A mechanism that does not rely solely in a change in the environmental pH is thus required for the release of pr from the mature particles upon release from infected cells. These results open up new avenues to understand the activation mechanism that yields mature, infectious YFV particles.

microbiology↗

bioRxiv2021

Single-cell analyses of immune thrombocytopenic patients reveal multiorgan dissemination of high-affinity autoreactive plasma cells

The major therapeutic goal for immune thrombocytopenia (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. 80% of patients possess anti-integrin IIb{beta}3 (GPIIbIIIa) IgG autoantibodies causing platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in ~50% of patients to recover a normal platelet count after anti-CD20 Rituximab-mediated B cell depletion and splenectomy suggest that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SC) reside in other anatomical compartments. We analyzed >3,300 single IgG-SC from spleen, bone marrow and/or blood of 27 patients with ITP revealing high inter-individual variability in affinity for GPIIbIIIa with variations over 3 logs. IgG-SC dissemination and range of affinities were however similar per patient. Longitudinal analysis of autoreactive IgG-SC upon treatment with anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-GPIIbIIIa IgG-SC in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

immunology↗

bioRxiv2021

The dengue virus NS1 protein conveys pro-inflammatory signals by docking onto human high-density lipoproteins

The nonstructural NS1 protein is a virulence factor secreted by dengue virus (DENV)-infected cells. NS1 is known to alter the complement system, activate immune cells and perturb endothelial barriers. Here we show that pro-inflammatory signals are triggered by a high affinity complex formed between NS1 and human high-density lipoproteins (HDL). Electron microscopy images of the NS1-HDL complexes show spherical HDL particles with rod-shaped NS1 protrusions on their surface. These complexes are readily detectable in the plasma of hospitalized dengue patients using anti-apolipoprotein A-I (ApoA-I) antibodies specific of the HDL moiety. The functional reprogramming of HDL particles by the NS1 protein as a means to exacerbate systemic inflammation during DENV infection provides a new paradigm linking the human lipoprotein network to dengue pathogenesis.

microbiology↗

bioRxiv2020

Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA

Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77[A]. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography and protein-protein interaction measurements - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA with an unprecedented level of detail. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Pol replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation phase and a processive phase during replication.

biochemistry↗

bioRxiv2019

Essential dynamic interdependence of FtsZ and SepF for Z-ring and septum formation in Corynebacterium glutamicum

The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, one of the largest bacterial phyla that includes the deadly human pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors as well as all positive and negative Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to directly interact with the conserved C-terminal tail of FtsZ but whose actual mode of action in cytokinesis is yet to be elucidated. We used a mechanistic cell biology approach to unveil the essential interdependence of FtsZ and SepF required for the formation of a functional Z-ring in the actinobacterial model organism C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and a reversible oligomerization interface regulated via an alpha helical switch. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, leading to a complex dynamic role of the protein at the division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.

microbiology↗