Conserved waters mediate structural and functional activation of family A (rhodopsin-like) G protein-coupled receptors

Abstract

G protein-coupled receptors with seven transmembrane ␣-helices (GPCRs) comprise the largest receptor superfamily and are involved in detecting a wide variety of extracellular stimuli. The availability of high-resolution crystal structures of five prototypical GPCRs, bovine and squid rhodopsin, engineered A 2A-adenosine, ␤1-and ␤2-adrenergic receptors, permits comparative analysis of features common to these and likely all GPCRs. We provide an analysis of the distribution of water molecules in the transmembrane region of these GPCR structures and find conserved contacts with microdomains demonstrated to be involved in receptor activation. Colocalization of water molecules associating with highly conserved and functionally important residues in several of these GPCR crystal structures supports the notion that these waters are likely to be as important to proper receptor function as the conserved residues. Moreover, in the absence of large conformational changes in rhodopsin after photoactivation, we propose that ordered waters contribute to the functional plasticity needed to transmit activation signals from the retinal-binding pocket to the cytoplasmic face of rhodopsin and that fundamental features of the mechanism of activation, involving these conserved waters, are shared by many if not all family A receptors.adrenergic receptor ͉ allosteric modulator ͉ ordered waters ͉ transmembrane proteins ͉ x-ray G protein-coupled receptors (GPCRs) are membrane proteins that mediate signal transduction by recognizing a wide range of extracellular stimuli ranging from photons of light, biogenic amines and odorants, to small peptides that all induce receptor activation. Activated GPCRs signal through heterotrimeric G proteins to activate effector enzymes, resulting in rapid signal amplification. Given the new-found wealth of structural information available from determination of several GPCR crystal structures, our purpose here is to provide a structural analysis of components likely to participate in the allosteric communication between the ligand-binding site near the extracellular domain and the cytoplasmic site involved in G protein coupling and activation.The first high-resolution crystal structure of a GPCR was that of bovine rhodopsin (1), an advance that facilitated the successful determination of other high-resolution bovine rhodopsin structures (2-5). One paradigm for GPCR activation involves large rigid body movements of up to 15 Å of the heptahelical transmembrane bundle upon receptor transition from an inactive to an active conformation (6). This notion has been challenged because large changes in these helices were not observed in the crystal structure of a late photointermediate of rhodopsin that retained the fullagonist, all-trans-retinylidene, and exhibited spectral features consistent with the metarhodopsin (Meta) II activated state (7). Newer studies using double electron-electron resonance (DEER) spectroscopy revised the predicted structural changes upon activation to be no more than 5 Å between helices (...

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

“…These lysines are covalently linked to the retinal chromophore. As with microbial 7TMRs, consensus clusters of buried water molecules are found in rhodopsin and ligandbinding GPCRs, consistent with the recent finding that family A GPCRs contain structurally conserved water molecules that mediate structural and functional activation (24). Relative to microbial 7TMRs, rhodopsin and ligand-binding GPCRs contain additional clusters of buried ionizable groups that correspond to the D/ERY (Asp/Glu-Arg-Tyr) motif, a conserved feature that couples GPCRs to G proteins.…”

“…Therefore we wondered whether GPCRs also contain buried electrostatic networks that might regulate G protein activation. Indeed, several studies have highlighted the prevalence of ionizable residues, water molecules (24), and ions such as sodium (25,26) in the cores of GPCRs. Here we used pHinder to analyze a comprehensive list of 27 rhodopsin and 70 ligandbinding GPCR structures.…”

Crystal structure of the ligand-free G-protein-coupled receptor opsin

“…It was also suggested that a proton relay via proton wires from the retinal binding pocket to the cytoplasmic surface (as shown in figure 4.6) may occur upon receptor activation [147].…”

Role of Bulk Water in Hydrolysis of the Rhodopsin Chromophore

“…Thus, ordered waters function as noncovalent cofactors that actively participate in transmitting the activation signal from the retinylidene-binding pocket to the cytoplasmic face of Rho, where binding of transducin occurs. These individual waters are observed in high resolution structures of Rho as well as other G protein-coupled receptors, and a high level of conservation of polar residues in close proximity to these waters is also found in all G proteincoupled receptor sequences (9,10).…”