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. KeywordsG protein-coupled receptors (GPCRs); NMR; three-dimensional structures; ligand recognition; receptor activation G protein-coupled receptors (GPCRs), also known as seven transmembrane-spanning receptors (7TMRs), are a large superfamily of signaling proteins expressed on the plasma membrane that function as receivers for extracellular stimuli [1]. About 1000 GPCRs have been identified in the human genome [2]. Since their signaling is involved in numerous physiological functions and pathological conditions, GPCRs constitute the molecular target of a significant percentage of the currently marketed drugs. Moreover, many additional members of the superfamily have been identified as potential targets for the treatment of a variety of diseases and are the object of substantial drug discovery efforts. From the molecular point of view, all GPCRs share a common molecular architecture, being composed of a single polypeptide chain folded into a bundle of seven α-helical transmembrane domains (TMs) connected by three extracellular and three intracellular loops (ELs and ILs). The N-terminus is located in the extracellular space, while the C-terminus is in the cytosol (Fig. (1)).Given that structure-based drug discovery is an efficient method to rationally design novel drugs and improve the properties of old drugs, the scientific community has been striving for a long time to shed light onto the elusive structure-function relationships of GPCRs employing a variety of direct biophysical and indirect biochemical methods [3]. The most direct method that has been used is X-ray crystallography. However, up until now, this technique has been *address for correspondence: stefanoc@mail.nih.gov. NIH Public Access Author ManuscriptCurr Pharm Des. Author manuscript; available in PMC 2010 January 5. Published in final edited form as:Curr Pharm Des. 2009 ; 15(35): 4003-4016. NIH-PA Author ManuscriptNIH-PA Author Manuscript...

Integrated Approach to Structure-Based Enzymatic Drug Design: Molecular Modeling, Spectroscopy, and Experimental Bioactivity

“…37 Computer-aided drug design forms part of the research strategy called structure-based design, which is an iterative combination of experimental and computer-based approaches. The results of both of these approaches can be combined to generate hypotheses and ideas (and test them) about the Table 2 Infrared spectroscopy a Signals from amide groups (CONH) in proteins are quite prominent and extensively used to determine structural information about proteins; phosphodiester groups are sensitive to hydrogen bonding; lipids can be studied through the ester group 1, 5, and 6 NMR spectroscopy Measures 1 H, 13 C, 15 N, and even some magnetic resonance signals of metal ions in biomolecules; it can provide structural information (through-space proton−proton interactions (NOE), backbone torsional angles, etc.) to study inhibitor/enzymes complexes; it provides solution-phase information and is a relatively fast technique 1, 3.2.2, 3.4, 5, and 6…”

Biophysical characterization of G-protein coupled receptor–peptide ligand bindingThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

“…Use of standard homonuclear and/or heteronuclear NMR spectroscopy (Wüthrich 1986, Cavanagh et al 2007) allows the determination of high-resolution structures of peptide ligands and, potentially, GPCRs (Tikhonova andCostanzi 2009, Gautier et al 2010). In the case of GPCR structure determination, most NMR studies to date have used the "divide and conquer" approach whereby fragments of a membrane protein are studied in isolation, improving feasibility of both protein production and spectroscopic characterization (general approach recently reviewed (Bordag and Keller 2010), and for GPCRs specifically (Yeagle and Albert 2007)).…”

“…In the case of GPCR structure determination, most NMR studies to date have used the "divide and conquer" approach whereby fragments of a membrane protein are studied in isolation, improving feasibility of both protein production and spectroscopic characterization (general approach recently reviewed (Bordag and Keller 2010), and for GPCRs specifically (Yeagle and Albert 2007)). Unfortunately, when studying sections of a GPCR in isolation, it becomes difficult to assign the tertiary structure of a protein since even if each segment gives an accurate secondary structure, the placement of each section in the receptor as a whole still needs to be determined by other methods (Yeagle et al 2001, Tikhonova andCostanzi 2009). Beyond structural determination, and often prior to the ability to determine a highresolution structure, NMR spectroscopy provides the capability to probe and characterize binding events at the level of individual amino acids in the ligand and/or receptor.…”

G-protein Coupled Receptors in Stem Cell Self-Renewal and Differentiation

“…The classical background on GPCRs and their signalling pathways has been covered extensively in a number of excellent recent reviews and therefore will only be briefly discussed here [15][16][17]. GPCRs are transmembrane receptors that consist of seven α-helical transmembrane domains separated by external and internal loops, with an intracellular C-terminal and an extracellular N-terminal.…”