Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets

Abstract

G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using an LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment, extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.

Figure 1

A. Attaching the syringe coupler to a gas-tight syringe. After the coupler is attached the syringe is filled with lipid. Then a second syringe, filled with protein solution, is attached to the other end of the coupler. B. Section through the syringe coupler.

Figure 2

Sequence of steps during manual set up of in meso crystallization trials. Reconstitution of protein in LCP (A – E) is described in section 3.1.2. Setting up trials in a glass sandwich plate (F – I) is described in section 3.3.2.

Figure 3

A. The LCP-FRAP station. B. Fluorescence recovery curves obtained for β₂AR-T4L reconstituted in monoolein based LCP after 12 hr incubation in the presence of 0.1 M Bis tris propane pH 7.0, 25 %(v/v) PEG 400, 5 %(v/v) 1,4-butanediol and different concentration of sodium sulfate: 0 M (triangles), 0.05 M (squares), 0.1 M (diamonds) and 0.4 M (circles).

Figure 4

Different ways of setting up crystallization trials in LCP: A. Microbatch, B. Sitting drop, C. Hanging drop, D. Modified hanging drop, E. Modified microbatch. In (D and E) LCP bolus (black) is sandwiched using a small 5 mm in diameter glass coverslip in order to improve optical properties and facilitate detection of crystals growing in LCP. The best conditions for detecting small colorless crystals are achieved when crystallization trials are performed in glass sandwich plates. (F – H) show top views of the glass sandwich plates used in our laboratory. Plates (F and G) are based on a standard microscope slide, which are convenient to use for setting up crystallization trials manually. Plate (H) has the SBS footprint and is suitable for both robotic and manual setups.

Figure 5

Needle-like crystals of the engineered adenosine A2a receptor grown in LCP. A. Bright-field illumination. B. With cross-polarizers. C. Fluorescence. The protein was trace-labeled with Cy 3 NHS. Fluorescence picture in (C) was recorded using excitation at 543 nm (22 nm bandwidth) and emission at 605 (60 nm bandwidth).

Figure 6

Sequence of steps illustrating opening of a well in the glass sandwich plate for crystal harvesting (see section 3.3.5.1. for details).