Biology subjects

Collu, G.

Publications and source records attributed to Collu, G..

5 recordsLinked to original sources

bioRxiv2021

Cryo-EM structure of a single-chain β1-adrenoceptor - AmpC β-lactamase fusion protein

The insertion of fusion proteins has enabled the crystallization of a wide range of G-protein-coupled receptors. Here, we adapted this engineering strategy to cryo-electron microscopy (cryo-EM). We inserted the soluble protein AmpC {beta}-lactamase into the third intracellular loop (ICL3) of ultra-thermostable {beta}1-adrenoceptor ({beta}1AR) via chimeric helix fusions. Biochemical and biophysical characterization showed that the resulting fusion protein after expression, solubilization and purification was monodisperse and able to bind the known {beta}1AR weak partial agonist cyanopindolol, and the antagonist propranolol. The protein particles comprised sufficient mass and discernable structural features to elucidate its cryo-EM structure in complex with cyanopindolol without any natural (G-proteins, arrestins) or artificial (Nanobodies, DARPins) binding partners, to an overall resolution of 4.2 [A]. The seven-helix architecture and helix eight, as well as both GPCR - AmpC {beta}-lactamase connections are clearly resolved. {beta}1AR is in its inactive conformation. 3D variability analysis revealed significant flexibility between the two protein domains and within the GPCR helices, offering insights into conformational dynamics. The map contains clear density for the cyanopindolol. The fusion protein geometry theoretically fits a wide range of class A GPCRs, presenting a powerful platform for structure elucidation of a diverse array of class A GPCR - ligand complexes by cryo-EM in the inactive receptor state. The approach furthermore holds potential for structure elucidation of GPCRs in the absence of ligands.

molecular biology↗

bioRxiv2020

Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology

Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible, which makes them unsuitable as structural building blocks. Here we show that the ER/K motif, a single -helical domain (SAH)1, can be seamlessly fused2 to terminal helices of proteins, forming an extended and partially free-standing rigid helix. Through the intrinsic stability of the SAH, two domains can be connected with a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano, and we show that a single SAH allows the connection of two separate structural domains with sufficient rigidity to form ordered crystals. The analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact in construct flexibility (for the study of protein dynamics), and to design novel biomaterials.

molecular biology↗

bioRxiv2020

Design, expression, purification and characterization of a YFP-tagged 2019-nCoV spike receptor-binding domain construct

2019-nCoV is the causative agent of the serious, still ongoing, worldwide COVID-19 pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFN2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA IMAC purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays.

biochemistry↗

bioRxiv2020

AT-527 is a potent in vitro replication inhibitor of SARS-CoV-2, the virus responsible for the COVID-19 pandemic

AT-527, an orally administered double prodrug of a guanosine nucleotide analog, has been shown previously to be highly efficacious and well tolerated in HCV-infected subjects. Herein we report the potent in vitro activity of AT-511, the free base form of AT-527, against several coronaviruses, including SARS-CoV-2, the causative agent of COVID-19. In normal human airway epithelial (HAE) cell preparations, the average concentration of AT-511 required to inhibit replication of SARS-CoV-2 by 90% (EC90) was 0.5 {micro}M, very similar to the EC90 for AT-511 against HCoV-229E, HCoV-OC43 and SARS-CoV in Huh-7 cells. No cytotoxicity was observed for AT-511 in any of the antiviral assays up to the highest concentration tested (100 {micro}M). Surprisingly, AT-511 was 30-fold less active against MERS-CoV. This differential activity may provide a clue to the apparent unique mechanism of action of the guanosine triphosphate analog formed from AT-527.

pharmacology and toxicology↗

bioRxiv2020

Inhibition of Wnt signalling by Notch via two distinct mechanisms

Notch and Wnt are two essential signalling pathways that help to shape animals during development and to sustain adult tissue homeostasis. Although, they are often active at the same time within a tissue, they typically have opposing effects on cell fate decisions. In fact, crosstalk between the two pathways is important in generating the great diversity of cell types that we find in metazoans. However, several different mechanisms have been proposed that allow Notch to limit Wnt signalling, driving a Notch-ON/Wnt-OFF state. Here we explore these different mechanisms in vertebrate cells and demonstrate two distinct mechanisms by which Notch itself, can limit the transcriptional activity of {beta}-catenin. At the membrane, independently of DSL ligands, Notch1 can antagonise {beta}-catenin activity through an endocytosis mediated mechanism that is dependent upon its interaction with Deltex and sequesters {beta}-catenin into the membrane fraction. Within the nucleus, the intracellular domain of Notch1 can also limit {beta}-catenin induced transcription through the formation of a complex that requires its interaction with RBPj{kappa}. We believe these mechanisms contribute to the robustness of cell-fate decisions by sharpening the distinction between opposing Notch/Wnt responses.

cell biology↗