Crystal structure of metarhodopsin II

摘要

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular hetero-trimeric G proteins (Gαβγ)~1. The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein2. Absorption of a photon causes retinal cisl trans isomerization and generates the agonist all- trans- retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Metall) is formed, in which the retinal Schiff base is still intact but depro-tonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplas-mic crevice for uptake of the interacting C terminus of the Gα subunit~(3-5). Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal's high concentration of opsin in an active conformation (Ops~*)~(6,7) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 A and 2.85 A crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Ga (GaCT2). GaCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Ga-derived peptide to Ops~* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all- trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.