Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

Abstract

Seven-transmembrane receptors (7TMRs) have evolved in prokaryotes and eukaryotes over hundreds of millions of years. Comparative structural analysis suggests that these receptors may share a remote evolutionary origin, despite their lack of sequence similarity. Here we used structure-based computations to compare 221 7TMRs from all domains of life. Unexpectedly, we discovered that these receptors contain spatially conserved networks of buried ionizable groups. In microbial 7TMRs these networks are used to pump ions across the cell membrane in response to light. In animal 7TMRs, which include light-and ligand-activated G protein-coupled receptors (GPCRs), homologous networks were found to be characteristic of activated receptor conformations. These networks are likely relevant to receptor function because they connect the ligandbinding pocket of the receptor to the nucleotide-binding pocket of the G protein. We propose that agonist and G protein binding facilitate the formation of these electrostatic networks and promote important structural rearrangements such as the displacement of transmembrane helix-6. We anticipate that robust classification of activated GPCR structures will aid the identification of ligands that target activated GPCR structural states.7-transmembrane receptor | G protein-coupled receptor | buried charge | structural bioinformatics | molecular evolution S even-transmembrane receptors (7TMRs) are present in all domains of life. In archaea and bacteria these receptors convert light energy into transmembrane ion gradients or intracellular signaling cascades. In humans 7TMRs comprise a family of more than 800 G protein-coupled receptors (GPCRs) that detect extracellular signals such as odorants, taste, light, hormones, and neurotransmitters. Although microbial and eukaryotic 7TMRs lack sequence similarity, their 3D folds share a degree of similarity that often is observed in remote evolutionary relationships identified by structure comparison algorithms (1).Recent breakthroughs in membrane protein crystallography have advanced our understanding of GPCR function by providing models of unactivated and activated receptor conformations (2-8). However, structure-based approaches for systematically quantifying differences between GPCR activation states are lacking. Here we introduce a computational approach that correlates GPCR activation with networks of electrostatic interactions in the receptor core. We show that membrane-spanning networks of ionizable residues, which we find are a common feature of microbial 7TMRs, also represent a unique signature of GPCR activation that is likely to be essential to receptor function.The molecular changes that accompany 7TMR activation were studied initially in bacteriorhodopsin and rhodopsin, the first microbial 7TMR and GPCR to be crystallized (9-11). More recently, a rapid expansion of GPCR structural information has revealed several molecular switches and structural features that are thought to be indicative of receptor activation. These include the ionic loc...

Structural basis of ligand interaction with atypical chemokine receptor 3

“…Many of these residues are buried in the TM domain of the receptor and cannot possibly be exposed to bulk solvent; therefore, their changes in oxidation are due to rearrangement of structured water molecules within the TM domain. Such water molecules are consistently found in several 7TM receptors by X-ray crystallography or structure-based MS experiments233839, and their positions appear to be affected by receptor activation, leading to restructuring of water-mediated polar residue networks404142. The path of ACKR3 residues with agonist-induced increases in oxidation rates is in striking agreement with solvent networks identified in active states of both rhodopsin and opioid receptors (Fig.…”

Regulation of Ras Paralog Thermostability by Networks of Buried Ionizable Groups

“…For this calculation we used an approach called consensus network analysis (CNA), which we previously developed to study the conservation of core charges in G protein-coupled receptors. 6 Using CNA, we aligned the pHinder-calculated core networks for each of the 885 Ras family GTPases. We then clustered the resultant set of 4389 core residues into distinct spatial nodes (Figure 2D).…”

“…7 When the G protein assembles with a receptor, a new core network is formed that links the GTP-binding pocket of Gα to the ligand binding pocket of the receptor. 6 …”

“…To that end, and as described here, we have implemented recently developed computational and experimental approaches to investigate the relationship between buried charge and protein thermostability. 6,7 Using a structure-based algorithm called pHinder, we show that networks of buried charge residues, hereafter referred to as core networks, are smaller in hyperthermophilic proteins than in mesophilic proteins. This finding led us to predict that proteins having greater thermostability should contain smaller core networks.…”

Thematic Minireview Series: New Directions in G Protein-coupled Receptor Pharmacology

“…Such structures would facilitate the design of biased agonists, both orthosteric and allosteric, that selectively promote coupling of receptors to G proteins, ␤-arrestins, receptor kinases, RGS proteins, and other GPCRs (as receptor oligomers). In the meantime, sophisticated computational techniques, including molecular dynamics simulations (6) and structural informatics analysis (7), are helping us to identify structural signatures and dynamic properties unique to activated receptors (3).…”