Biology subjects

Warne, T.

Publications and source records attributed to Warne, T..

3 recordsLinked to original sources

bioRxiv2024

Multimodal intrinsic activation of GPCRs in ultrastable plasma membrane nanodomains

G protein-coupled receptors (GPCRs) mediate many physiological functions and are key targets in drug development1-3. A long-held tenet of molecular pharmacology is that GPCRs can spontaneously sample preexisting active conformations. This concept is pivotal to our understanding of ligand pharmacology4, however, direct evidence supporting it has only been obtained with reconstituted receptors5-12. Here, we introduce a method for quantitatively imaging the intrinsic activation probability of GPCRs directly at the plasma membrane of live cells, utilizing fluorescent conformational biosensors13,14. Our findings unveil a remarkable spatial multimodality in intrinsic activation probability, with a significant majority (up to 99%) of plasma membrane-expressed receptors showing negligible spontaneous activation. In contrast, the remaining minority of receptors exhibits spontaneous activation up to 22-fold higher than previously estimated. Experiments and theoretical calculations revealed that receptors diffuse into and out of ultralong-lived ([~]5 minutes) nanodomains where the local membrane curvature allosterically enhances activation in the absence and presence of ligands. Extensive testing across five prototypic GPCRs indicates spatial nanoscale multimodality is ubiquitous, but varying in magnitude depending on the receptor and cell type. Upending conventional wisdom, this study reveals that drug efficacy is not a constant number but a spatiotemporal function {varepsilon} (x, y, z, t) whose properties define and multiplex the signaling potency and efficacy of ternary complexes of GPCRs and likely other plasma membrane-receptors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/582451v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e4680borg.highwire.dtl.DTLVardef@16aaebcorg.highwire.dtl.DTLVardef@f5df52org.highwire.dtl.DTLVardef@18a5ea_HPS_FORMAT_FIGEXP M_FIG C_FIG GPCR spontaneous activation and intrinsic efficacy are not uniform across the plasma membrane but exhibit ultralong-lived spatial multimodality. Spatial variations in the curvature and composition of the plasma membrane, lead to the emergence of ultralong-lived nanodomains with contrasting physicochemical properties that allosterically regulate GPCR conformations. This results in a multimodal landscape of intrinsic efficacy{epsilon} (x, y, z, t) that ultimately governs cell signaling. XY scalebar: 500 nm. Z-range: 100 nm.

biophysics↗

bioRxiv2020

Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

The {beta}1-adrenoceptor ({beta}1AR) is a G protein-coupled receptor (GPCR) activated by the hormone noradrenaline, resulting in the coupling of the heterotrimeric G protein Gs1. G protein-mediated signalling is terminated by phosphorylation of the receptor C-terminus and coupling of {beta}-arrestin 1 ({beta}arr1, also known as arrestin-2), which displaces Gs and induces signalling through the MAP kinase pathway2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins (biased agonism)3 is important in drug development, as the therapeutic effect may arise from only one signalling cascade, whilst the other pathway may mediate undesirable side effects4. To understand the molecular basis for arrestin coupling, we determined the electron cryo-microscopy (cryo-EM) structure of the {beta}1AR-{beta}arr1 complex in lipid nanodiscs bound to the biased agonist formoterol5, and the crystal structure of formoterol-bound {beta}1AR coupled to the G protein mimetic nanobody Nb806. {beta}arr1 couples to {beta}1AR in a distinct manner to how Gs couples to {beta}2AR7, with the finger loop of {beta}arr1 occupying a narrower cleft on the intracellular surface closer to transmembrane helix H7 than the C-terminal 5 helix of Gs. The conformation of the finger loop in {beta}arr1 is different from that adopted by the finger loop in visual arrestin when it couples to rhodopsin8, and its {beta}-turn configuration is reminiscent of the loop in Nb80 that inserts at the same position. {beta}1AR coupled to {beta}arr1 showed significant differences in structure compared to {beta}1AR coupled to Nb80, including an inward movement of extracellular loop 3 (ECL3) and the cytoplasmic ends of H5 and H6. In the orthosteric binding site there was also weakening of interactions between formoterol and the residues Ser2115.42 and Ser2155.46, and a reduction in affinity of formoterol for the {beta}1AR-{beta}arr1 complex compared to {beta}1AR coupled to mini-Gs. These differences provide a foundation for the development of small molecules that could bias signalling in the {beta}-adrenoceptors.

molecular biology↗

bioRxiv2018

Molecular basis for high affinity agonist binding in GPCRs

A characteristic of GPCRs in the G protein-coupled state is that the affinity of the agonist often increases significantly, but the molecular basis for this is unclear. We have determined six active-state structures of the {beta}1-adrenoceptor ({beta}1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. A direct comparison with structures of {beta}1AR in inactive states bound to the identical ligands showed a 24-42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. GPCRs are highly conserved, so these factors will likely be essential in increasing the affinity of a wide range of structurally distinct agonists.\n\nOne Sentence SummaryHigh affinity agonist binding to G protein-coupled GPCRs results from an increase in the number and strength of protein-ligand interactions.

biochemistry↗