Prediction of the 3-D structure of rat MrgA G protein-coupled receptor and identification of its binding site

Abstract

Mrg receptors are orphan G protein-coupled receptors (GPCRs) located mainly at the specific set of sensory neurons in the dorsal root ganglia, suggesting a role in nociception. We report here the 3-D structure of rat MrgA (rMrgA) receptor [obtained from homology modeling to the recently validated predicted structures of mouse MrgA1 and MrgC11] and the structure of adenine (a known agonist, K i =18nM) bound to rMrgA. This predicted binding site is located within transmembrane helical domains (TMs) 3, 4, 5 and 6, with Asn residues in TM3 and TM4 identified as the key residues for adenine binding. Here the side chain of Asn88 (TM3) forms two pairs of hydrogen bonds with N3 and N9 of adenine while Asn146 (TM4) makes two pairs of hydrogen bonds with N1 and N6 of adenine. These interactions lock adenine tightly in the binding pocket. We also predict the binding site of guanine (not an agonist) and seven other derivatives. Guanine cannot make the hydrogen bond to Asn146 (TM4), leading to binding too weak to be observed experimentally. The predicted binding affinity for other adenine derivatives correlates with the availability of the hydrogen bonds to these two Asn residues. These results validate the predicted structure for rat MrgA and suggest mutation experiments that could further validate the structure. Moreover the predicted structure and binding site should be useful for seeking other small molecule agonists and antagonists.

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

“…For mutagenesis studies, we selected certain amino acids predicted to potentially participate in ligand binding or receptor activation based on (a) sequence comparison looking for conserved residues, (b) predictions made by Heo et al [15] in 2007 based on a homology model, and (c) our own, refined homology model incorporating recent progress in GPCR structure elucidation [29]. X-ray structures for a number of the more prominent class A GPCRs have recently become available [29,31], which can serve as templates for homology modeling.…”

“…This sequence may represent a potential signal peptide. Therefore, the numbering used in the present study, which is based on the whole coding sequence including the N-terminal putative signal peptide (AJ311952), differs from the numbering used by Heo et al by 27 amino acids [15]. We additionally provide the Ballesteros-Weinstein numbering: to the most conserved amino acid residue in each TMD, the number 50 is assigned, and numbers for other amino acid residues are decreased towards the N-terminus and increased towards the Cterminus; the thus created amino acid number is preceded by the helix number [28].…”

“…Heo et al had published a homology model of the rAdeR based on homology models of the mouse MrgA1 and MrgC11 receptors [15]. The rAdeR sequence that they used is lacking 27 amino acids at the N-terminus.…”

“…In addition, the highly conserved cysteine residue in the ECL2 gets the position number 45.50, and amino acids located in the ECL2 are numbered in relation to Cys 45.50 . According to the prediction by Heo et al, the binding pocket is located between helices 3, 4, 5, and 6, and adenine is predicted to mainly interact with five amino acids: Phe110 3.24 (Phe83 [15], TMD3), Asn115 3.29 (Asn88 [15], TMD3), Asn173 4.60 (Asn146 [15], TMD4), Leu201 5.47 (Leu174 [15], TMD5), and His252 6.54 (His225 [15], TMD6), of which the two asparagine residues Asn 3.29 and Asn4.60 were hypothesized to be most important [15].…”

“…Based on a homology model of the rAdeR previously published by Heo et al [15], and a refined model developed in our group, we replaced the amino acid residues predicted to be involved in adenine binding by alanine. The mutant receptors were subsequently analyzed in radioligand binding and functional studies in comparison to the wt receptor.…”