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Crystal structure of pre-activated arrestin p44
Kim, Yong Ju,Hofmann, Klaus Peter,Ernst, Oliver P.,Scheerer, Patrick,Choe, Hui-Woog,Sommer, Martha E. Nature Publishing Group, a division of Macmillan P 2013 Nature Vol.497 No.7447
Arrestins interact with G-protein-coupled receptors (GPCRs) to block interaction with G proteins and initiate G-protein-independent signalling. Arrestins have a bi-lobed structure that is stabilized by a long carboxy-terminal tail (C-tail), and displacement of the C-tail by receptor-attached phosphates activates arrestins for binding active GPCRs. Structures of the inactive state of arrestin are available, but it is not known how C-tail displacement activates arrestin for receptor coupling. Here we present a 3.0 Å crystal structure of the bovine arrestin-1 splice variant p44, in which the activation step is mimicked by C-tail truncation. The structure of this pre-activated arrestin is profoundly different from the basal state and gives insight into the activation mechanism. p44 displays breakage of the central polar core and other interlobe hydrogen-bond networks, leading to a ∼21° rotation of the two lobes as compared to basal arrestin-1. Rearrangements in key receptor-binding loops in the central crest region include the finger loop, loop 139 (refs 8, 10, 11) and the sequence Asp 296–Asn 305 (or gate loop), here identified as controlling the polar core. We verified the role of these conformational alterations in arrestin activation and receptor binding by site-directed fluorescence spectroscopy. The data indicate a mechanism for arrestin activation in which C-tail displacement releases critical central-crest loops from restricted to extended receptor-interacting conformations. In parallel, increased flexibility between the two lobes facilitates a proper fitting of arrestin to the active receptor surface. Our results provide a snapshot of an arrestin ready to bind the active receptor, and give an insight into the role of naturally occurring truncated arrestins in the visual system.
Crystal structure of metarhodopsin II
Choe, Hui-Woog,Kim, Yong Ju,Park, Jung Hee,Morizumi, Takefumi,Pai, Emil F.,Krau?, Norbert,Hofmann, Klaus Peter,Scheerer, Patrick,Ernst, Oliver P. Nature Publishing Group, a division of Macmillan P 2011 Nature Vol.471 No.7340
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 heterotrimeric G proteins (G慣棺款). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta??II) is formed, in which the retinal Schiff base is still intact but deprotonated. 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 cytoplasmic crevice for uptake of the interacting C terminus of the G慣 subunit. 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*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0??? and 2.85??? crystal structures, respectively, of Meta??II alone or in complex with an 11-amino-acid C-terminal fragment derived from G慣 (G慣CT2). G慣CT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar G慣-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.