Ice breaking in GPCR structural biology

Abstract

G-protein-coupled receptors (GPCRs) are one of the most challenging targets in structural biology. To successfully solve a high-resolution GPCR structure, several experimental obstacles must be overcome, including expression, extraction, purification, and crystallization. As a result, there are only a handful of unique structures reported from this protein superfamily, which consists of over 800 members. In the past few years, however, there has been an increase in the amount of solved GPCR structures, and a few high-impact structures have been determined: the peptide receptor CXCR4, the agonist bound receptors, and the GPCR-G protein complex. The dramatic progress in GPCR structural studies is not due to the development of any single technique, but a combination of new techniques, new tools and new concepts. Here, we summarize the progress made for GPCR expression, purification, and crystallization, and we highlight the technical advances that will facilitate the future determination of GPCR structures.

Figure 1

Structural models of the TM4-3-5 interface, indicating the most frequent 3.41 (Ballesteros and Weinstein System) mutation in GPCR structures. (A, B) Rhodopsin inactive state structure (PDB ID code 1U19) and β₂AR structure showing residues proximal to 3.41. TM helices are colored grey and side-chains carbon atoms are colored green. (C) Clustal W sequence alignment illustrating the residue conservation in TM3, TM4, and TM5 for the βARs, rhodopsin and several members of the biogenic amine family. Identical residues are highlighted in grey. Key residues mentioned in the text are marked with asterisks. Position 3.41 is highlighted in yellow.

Figure 2

A snake β₂AR plot to brief summary of the mutagenesis studies aiming higher GPCR stability^{, , , , ,} . The mutations on different receptors are applied to β₂AR receptor based on the Ballesteros and Weinstein Number System and labeled in red on the Figure. The post translational modifications such as glycosylation and phosphorylation are labeled as indicated.

Figure 3

A brief summary of the new detergents to facilitate the GPCR structure study. (A) MNG; (B) branched detergent; (C) tandem facial amphiphiles (TFA); (D) facial amphiphiles; (E) a cartoon about the working hypothesis of facial amphiphiles and tandem facial amphiphiles in compare with conventional detergents.

Figure 4

Phase diagram of lipidic cubic phase use Monoolein. A) A representative figure of GPCR in bicelle. The receptor is surrounded by a DMPC lipid bilayer, which is further covered by detergents. The receptor-lipid-detergent is afloat in the solution. D) the phase-temp diagram of Monoolein and water system, the phase will transit as lipid:water ratio or temperature change; C) crystallization of GPCRs in cubic phase, the receptor is in cartoon, the bicontinuous lipid bilayer is drawn in yellow and white and the dark blue represents the water channel and aqueous solution; D) Phase behavior of lipid-water-PEG400 system, representing the influences of additives and lipids on the phase.