Biology subjects

Bathgate, R. A. D.

Publications and source records attributed to Bathgate, R. A. D..

4 recordsLinked to original sources

bioRxiv2022

The dynamic nature of neurotensin receptor 1 (NTS1) allostery and signaling bias

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13C{varepsilon}H3-methionine NMR spectroscopy to show that phosphatidylinositol-4,5-bisphosphate (PIP2) promotes transducer complexation not by dramatically altering the receptor structure but by strengthening long-range allosteric connections, in the form of correlated conformational kinetics, between the orthosteric pocket and highly-conserved activation motifs. {beta}-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on the rate. A {beta}-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

biochemistry↗

bioRxiv2022

A unique peptide recognition mechanism by the human relaxin family peptide receptor 4 (RXFP4)

Members of the insulin superfamily regulate a variety of biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs), which are class A G protein-coupled receptors (GPCRs), to exert pleiotropic actions. Here, we report three cryo-electron microscopy structures of RXFP4-Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053, both were discovered through medicinal chemistry efforts. The B chain of INSL5 adopts a single -helix that penetrates into the orthostatic pocket, while the A chain sits above the orthosteric pocket to interact with the extracellular surface of RXFP4, revealing a unique peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. DC591053 selectively mimicked the action of INSL5 at RXFP4 whereas compound 4 activated both RXFP3 and RXFP4. Comparison of peptide binding modes within the insulin superfamily displayed diverse interaction mechanisms distinct to each type of the peptides. Our findings not only provide valuable insights into ligand recognition and subtype selectivity among class A GPCRs, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

molecular biology↗

bioRxiv2021

Structural insights into the modes of relaxin-binding and tethered-agonist activation of RXFP1 and RXFP2

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signalling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.

biophysics↗

bioRxiv2019

α1A-adrenoceptor inverse agonists and agonists modulate receptor signalling through a conformational selection mechanism

G-Protein Coupled Receptors (GPCRs) transmit signals across the cell membrane via an allosteric network from the ligand-binding site to the G-protein binding site via a series of conserved microswitches. Crystal structures of GPCRs provide snapshots of inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here we analyse the correlation between ligand binding and receptor conformation of the 1A-adrenoceptor, known for stimulating smooth muscle contraction in response to binding noradrenaline. NMR of 13C{varepsilon}H3-methionine labelled 1A-adrenoreceptor mutants, each exhibiting differing signalling capacities, revealed how different classes of ligands modulate receptor conformational equilibria. 13C{varepsilon}H3-methionine residues near the microswitches revealed distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling between the microswitches controls receptor conformation and underlies the mechanism of ligand modulation of GPCR signalling in cells.

biochemistry↗