Crystal structure of pre-activated arrestin p44

Abstract

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.

Spatial and Temporal Aspects of Signaling by G-Protein–Coupled Receptors

“…In the arrestin case, an X-ray structure of b-arrestin bound to a b 2 -adrenergic receptor phosphopeptide has been obtained, in which the stabilizing C-terminal tail of arrestin is displaced (Shukla et al, 2013). The structure is similar to the one of a preactivated C-terminally truncated arrestin-1 (p44 protein) (Kim et al, 2013). Although a direct comparison between G-protein and arrestin precomplexes is not yet possible, a partial structuring of a key interaction site (GaCT fragment or Arr-FL finger loop) compared with the basal state appears to be a common property of the precomplexes.…”

Functional map of arrestin-1 at single amino acid resolution

“…Among those are Lys14, Lys15, and Lys110 close to the three-element interaction site. Another one is Lys300 in the gate loop (residues Asp296-Asn305) that reorganizes during breakage of the polar core (9). The weakest binder revealed by our screen, Arg29, is located in the center of this positively charged region.…”

“…Phosphosensing mechanism in arrestin-1. Binding of arrestin mutants (blue and ribbon width indicate residues which increase binding to P-ROS* upon mutation; red indicates residues which decrease binding to P-ROS* upon mutation) plotted on the crystal structures of inactive (8), preactivated p44 arrestin (9), and a homology model of arrestin-1 based on the crystal structure of arrestin-2 bound to a receptor phosphopetide (10). Several residues including three hydrophobic phenylalanines (Phe375, Phe377, and Phe380) and the charged Arg382 anchor the C tail of arrestin-1 into the three-element interaction and polar core regulatory sites (Left).…”

Conformational Selection and Equilibrium Governs the Ability of Retinals to Bind Opsin

“…2 and under "Experimental Procedures." Coordinates from Protein Data Bank codes 3DQB and 4PXF were used for G t C-term and Arr peptide-bound opsin structures, respectively (17,56), and codes 3SN6 and 4J2Q were used for G protein and arrestin models, respectively (45,86).…”