Generic GPCR residue numbers - aligning topology maps while minding the gaps

Abstract

Generic residue numbers facilitate comparisons of, for example, mutational effects, ligand interactions, and structural motifs. The numbering scheme by Ballesteros and Weinstein for residues within the class A GPCRs (G protein-coupled receptors) has more than 1100 citations, and the recent crystal structures for classes B, C, and F now call for a community consensus in residue numbering within and across these classes. Furthermore, the structural era has uncovered helix bulges and constrictions that offset the generic residue numbers. The use of generic residue numbers depends on convenient access by pharmacologists, chemists, and structural biologists. We review the generic residue numbering schemes for each GPCR class, as well as a complementary structure-based scheme, and provide illustrative examples and GPCR database (GPCRDB) web tools to number any receptor sequence or structure.

Keywords: G protein-coupled receptor; ligand binding; mutational effects; sequence alignments; structural motifs.

Figure 1. Class A/B common receptor activation motif in TM7

Intracellular view of structural superposition of CRF₁ (magenta, PDB: 4K5Y), GCGR (cyan, PDB: 4L6R) and β₂AR (yellow, PDB: 3SN6) receptors. The conserved Tyr residue Y^7.53a.57b and in CRF1₁ the thermostabilizing mutation Y363^7.57bA are shown in stick representation. In GCGR the polar interactions of Y400^7.57b with T351^6.42b and E245^3.50b residues are represented as dotted lines.

Figure 2. Class A/C unique features of conserved residues in TM6 involved in ligand binding and receptor activation

Side view of Rhodopsin (purple, PDB: 2X72) and mGluR1 (green, PDB: 4OR2). 11-cis-retinal and FITM are shown in line representation. L^5.44c and the aromatic residues W^6.48a.50c and Y/F^6.51a.53c are shown in stick representation. In mGluR 1 the polar interaction between W^6.50c and L^5.44c is represented as a dotted line.

Figure 3. Bulges and constrictions in GPCR class A, B, C and F crystal structures

Bulged (A–I) and constricted (J–O) helical turns have one extra and lacking residue position, respectively, when superposed to an undistorted helix reference. The structural and sequence alignments of equivalent residues therefor contain a gap, which offsets all following generic residue numbers (grey). The GPCRDB generic residue numbers (black) are structure-based and accounts for bulges and constrictions by numbering an extra residue as the preceding followed by a 1, e.g. 1.40×411, and a skipping a lacking position. To facilitate cross-class comparisons, their alignments have been assigned GPCRDB numbers for all classes. The percentages to the lower right of the structures represent the conservation/frequency of the bulge or constriction within the class, as seen in the GPCRDB alignments [25]. A_2A has two bulges in TM5 (D) giving a double offset, e.g. 5.39 vs. 5×41. Spheres represent the alpha carbon of each residue. Receptors; 5-HT_2B (PDB: 4IB4), A_2A (PDB: 4EIY), β₁AR (PDB: 4AMJ), β₂AR (PDB: 2RH1), CRF₁ (PDB: 4K5Y), H₁ (PDB: 3RZE), P2Y₁₂ (PDB: 4NTJ), S1P₁ (PDB: 3V2Y), SMO (PDB: 4JKV) and mGlu₁ (PDB: 4OR2).

Figure 4. TM bulges and constrictions orient different residue numbers in the ligand binding site in GPCR crystal structures

Comparison of the T^2.56XP^2.58 stabilized region (orange) in TM2 of the CXCR4 chemokine receptor that orients W^2.60 and D^2.63 into the 1T1t binding site [62] (A), and the alpha-bulge in TM2 of the turkey β₁ adrenergic receptor that orients G^2.61×60 and L^2.64×63 into the carvedilol binding site [68] (B). Comparison of the constricted region (orange) in TM4 of the histamine H₁ receptor [70] that orients W^4.56×57 into the doxepin binding site (C), and TM4 of the M₂ muscarinic receptor [72] that orients W^4.57 into the 3-quinuclidinyl-benzilate binding site (D). Ligand carbon atoms are colored magenta. Residues W^6.48 and Y/F^6.51 in TM6 are shown as reference (note that TM6 in panels A-B is rotated ~120 degrees compared to TM6 in panels C–D).

Figure 5. GPCRDB Residue numbering tools

GPCRDB offers a suite of residue numbering tools. A) Sequence alignments are gapped to account for bulges and constrictions and list both the structure-based (GPCRDB) and sequence-based residue numbers, for each class. The example, obtain from the structure browser tool, shows the TM2 alignment for two crystallized class A-C receptors and one class F GPCR. B) Lookup tables show generic and receptor-specific residue numbers and can be used to compare the equivalent receptor residues. The example covers TM1 for the class A receptors included in the case stories. C) Structures can be browsed or uploaded in pdb format to assign up to two generic numbering schemes to the transmembrane residues. The example shows the carazolol binding site of the β2-adrenergic receptor (PDB: 2RH1).