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

Abstract

G protein-coupled receptors with seven transmembrane alpha-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, beta(1)- and beta(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.

Fig. 1.

Structural superpositioning diagram of high-resolution crystal structures of bovine rhodopsin (red), squid rhodopsin (wheat), mutant β₁-adrenergic receptor (light blue), mutant β₂-adrenergic receptor (navy blue), and bovine opsin (gray) demonstrating a high level of overall structural similarity. Also shown are those water molecules that colocalize in the transmembrane helices.

Fig. 2.

Water molecules observed in the crystal structures of family A GPCRs. Structural superpositioning of the C^α chains reveals that many of the ordered waters found in available high-resolution GPCR crystal structures are colocalized. (A) Positions of the waters (shown as spheres) from the structures of bovine rhodopsin (red), squid rhodopsin (wheat), bovine opsin (light gray), bovine opsin with peptide (dark gray), mutant β₁-adrenergic receptor (light blue), mutant β₂-adrenergic receptor (navy blue), and A_2A-adenosine receptor (orange) after superpositioning each GPCR structure with that of bovine rhodopsin. Waters are localized to 15 regions within the helical bundles of these GPCRs, shown here in the context of the structure of rhodopsin. (B) Water cluster conserved between squid rhodopsin, mutant β₂-adrenergic receptor and bovine rhodopsin. Waters 2, 3, and 4 notably make contacts with D^2.50 and Y^7.53 and are within hydrogen-bonding distance of highly conserved residues in H2, H6, and H7. Waters 11, 12, and 13 are part of an extended network of waters that may constitute the communication pathway from regions adjacent to the ligand-binding pocket to the cytoplasmic face of the receptors. (C) Water 6, unique to bovine rhodopsin, interacts with the Schiff base counterion E113; this water potentially plays a role in Schiff base stabilization and proton transfer during photoactivation and interconversion between MI and MII. (D) Water cluster 9, found in 5 of the 6 GPCR structures near the highly conserved WxP^6.50F/Y motif.

Fig. 3.

Conservation of hydrogen-bonding networks. Colocalized waters present in the crystal structures of bovine rhodopsin (A), squid rhodopsin (B), mutant β₂-adrenergic receptor (C), and A_2A-adenosine receptor (D) make contact with highly conserved amino acid residues. The lines connecting waters and helices indicate putative hydrogen bonds. For water clusters, yellow circles indicate conserved networks shared by all receptors, gray circles indicate interactions shared among squid rhodopsin, mutant β₂-adrenergic, and A_2A-adrenergic receptor, black circles indicate conserved networks shared among bovine rhodopsin, squid rhodopsin, and mutant β₂-adrenergic receptor, red circles indicate conserved networks shared between bovine rhodopsin and mutant A_2A-adenosine receptor, and finally, orange circles indicate conserved networks shared between squid rhodopsin and mutant β₂-adrenergic receptor.