Crystallization of G protein-coupled receptors

Abstract

Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor-receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the β2-adrenergic receptor (β2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For β2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a β2-AR/T4-lysozyme chimera bound to the antagonist carazolol.

Keywords: Ammonium sulfate; Crystallization; G protein-coupled receptor; Lipidic cubic phase; Purification; Rhodopsin; β2-Adrenergic receptor.

FIGURE 24.1

Immunoaffinity purification of bovine rhodopsin from ROS solubilized in NG. (A) Purified rhodopsin was eluted from a 1.5 cm×30 cm column containing 46 mL of 1D4-Sepharose. (B) Absorption spectrum of purified rhodopsin (an aliquot was diluted in UV buffer). (C) Coomassie-stained SDS-PAGE gel of purified rhodopsin. MW markers (SeeBlue^® Plus2, Invitrogen) are shown in the right lane.

FIGURE 24.2

(NH₄)₂SO₄-induced phase separation of purified rhodopsin. (A) Solid (NH₄)₂SO₄ was added to rhodopsin purified in 25 mM NG (■), 50 mM NG (●), or 50 mM OG (□). [Rho]_pool corresponds to the concentration of rhodopsin prior to treatment with (NH₄)₂SO₄, and [Rho]_conc is the final concentration of rhodopsin after (NH₄)₂SO₄-induced phase separation. Modified and expanded from Salom, Le Trong, et al. (2006). (B) Phase separation in solutions containing 1% detergent in 100 mM MES, pH 6.35, after adding saturating amounts of solid (NH₄)₂SO₄ and overnight incubation on ice (HG, heptyl glucoside; HTG, heptyl-thio- glucoside; OG, octyl glucoside; OTG, octyl-thio-glucoside; OGNG, octyl glucoside neopentyl glycol; NG, nonyl glucoside).

FIGURE 24.3

Photoactivated rhodopsin crystals surrounding a colorless (NH₄)₂SO₄ crystal.

FIGURE 24.4

Crystal structure of β2-AR(E122W)/T4L. (A) SDS-PAGE analysis of a purified β2-AR(E122W)/ T4L sample developed with silver staining. Both the purified monomer and dimer are present. (B, C) Representative crystals imaged under crossed polarizers. Inset in panel B shows a close-up of microcrystals. (B) Crystals grown in 0.1 M HEPES, pH 7.0, 0.15 M ammonium fluoride, 30% PEG 400, and 7% 1,4-butanediol. (C) Crystals grown in 0.1 M HEPES, pH 7.0, 0.15 M sodium sulfate, 30% PEG 400, and 7% 1,4-butanediol. (D) Representative X-ray diffraction pattern from a microcrystal with dimensions of 20×10×5 μm. (E) A part of electron density of β2-AR(E122W)/T4L crystals.