Unraveling the structure and function of G protein-coupled receptors through NMR spectroscopy

Abstract

G protein-coupled receptors (GPCRs) are a large superfamily of signaling proteins expressed on the plasma membrane. They are involved in a wide range of physiological processes and, therefore, are exploited as drug targets in a multitude of therapeutic areas. In this extent, knowledge of structural and functional properties of GPCRs may greatly facilitate rational design of modulator compounds. Solution and solid-state nuclear magnetic resonance (NMR) spectroscopy represents a powerful method to gather atomistic insights into protein structure and dynamics. In spite of the difficulties inherent the solution of the structure of membrane proteins through NMR, these methods have been successfully applied, sometimes in combination with molecular modeling, to the determination of the structure of GPCR fragments, the mapping of receptor-ligand interactions, and the study of the conformational changes associated with the activation of the receptors. In this review, we provide a summary of the NMR contributions to the study of the structure and function of GPCRs, also in light of the published crystal structures.

Figure 1

The topology of G protein coupled receptors.

Figure 2

Structural alignment of the NMR coordinates of GPCR portions deposited in the PDB and the crystal structures of the and bovine rhodopsin. Panel A: superimposition of the β₂-AR crystal structure (2RH1, red) with the NMR-derived structures of the N-termini of PTH₁ (1BL1–white) and CCK₁ (1D6G, orange), EL1 of S₁P₄, (2DCO, aquamarine), IL3 of CB₁ (1LVQ, light blue), TM6 of Ste2pR (1PJD, yellow), and EL3 of CCK₁ (1HZN, pink) and CCK₂ (1L4T, dark blue). Panel B: superimposition of the crystal structure of the β₁-AR (2VT4, plum) with the NMR-derived structure of H8 of the same receptor (1DEP, green). Panel C: superimposition of the crystal structure of rhodopsin (2HPY, white) and the NMR-derived structure of the C-terminus of the same receptor (1NZS, blue purple). Pictures prepared with Maestro 8.0.308, Schrodinger.

Figure 3

Superimposition of the crystal structures and the NMR-based three-dimensional models of bovine rhodopsin. Panel A: Superimposition of the X-ray structure (1GZM, white) and the NMR-based model of the ground state (1LN6, purple) receptor. Panel B: Superimposition of the X-ray structure of opsin in its G-protein-interacting conformation (3DQB, orange) and NMR-based model of Meta II rhodopsin (1JFP, green). Picture prepared with Maestro 8.0.308, Schrodinger.

Figure 4

Two dimensional schematic representation of the retinal binding site in ground state rhodopsin. Picture prepared with MOE 2008.10, Chemical Computing Group.

Figure 5

Panel A: Positions of the ligand atoms and residues labeled in order to study the activation-related conformational changes of rhodopsin. The retinal atoms (C5, C9, C11, C13, C14, C15, C19, C20) are in purple; residues 114, 118, 121, 178, 188, 191, 196, and 268 are in green; residue 265 is in blue; residues 122 and 211 are in pink; the residues mutated to Cys to be labeled with trifluoroethylthio groups are in yellow. For simplicity, only one carbon of the residue backbone is shown. Panel B: All Trp and Lys residues of rhodopsin. Picture prepared with Maestro 8.0.308, Schrodinger.