Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin

Abstract

Arrestins regulate signaling and trafficking of G protein-coupled receptors by virtue of their preferential binding to the phosphorylated active form of the receptor. To identify sites in arrestin involved in receptor interaction, a nitroxide-containing side chain was introduced at each of 28 different positions in visual arrestin, and the dynamics of the side chain was used to monitor arrestin interaction with phosphorylated forms of its cognate receptor, rhodopsin. At physiological concentrations, visual arrestin associates with both inactive dark phosphorylated rhodopsin (P-Rh) and light-activated phosphorylated rhodopsin (P-Rh*). Residues distributed over the concave surfaces of the two arrestin domains are involved in weak interactions with both states of phosphorhodopsin, and the flexible C-terminal sequence (C-tail) of arrestin becomes dynamically disordered in both complexes. A large-scale movement of the C-tail is demonstrated by direct distance measurements using a doubly labeled arrestin with one nitroxide in the C-tail and the other in the N-domain. Despite some overlap, the molecular "footprint" of arrestin bound to P-Rh and P-Rh* is different, showing the structure of the complexes to be unique. Strong immobilizing interactions with residues in a highly flexible loop between beta-strands V and VI are only observed in complex with the activated state. This result identifies this loop as a key recognition site in the arrestin-P-Rh* complex and supports the view that flexible sequences are key elements in protein-protein interactions.

Conformation of receptor-bound visual arrestin

“…2A and Fig. S3), consistent with a more restricted motion for this loop at a direct receptor interaction site (11).…”

“…EPR spectra of 139R1 suggested that this loop has high mobility in both the basal and active states (11), consistent with the relatively broad distributions for distances between 139R1 and R1 at reference sites 60, 173, 251, and 267, both in the absence and presence of P-Rh*. All distance changes are consistent with a movement of the 139 loop away from the finger loop, which apparently resides at the receptor-binding surface (11). We propose that the observed large movement of the 139 loop facilitates receptor binding involving the finger loop and adjacent elements.…”

“…In addition, the top Rosetta model shows that the finger loop can adopt an α-helical conformation in the solution tetramer, in contrast to the crystal, where it is disordered or unresolved (4). Previously reported EPR spectra of R1 in positions 72 and 74 (11) in the finger loop show at least two components, suggesting a possibility of at least two distinct conformational states. Taken together, these results suggest that the finger loop may exist in multiple conformations in equilibrium.…”

“…Arrestin-1 was proposed to undergo a conformational rearrangement during the P-Rh* interaction that results in the release of the C-terminal sequence (C tail) (8, 9) but does not involve major secondary structure changes (8, 10). Recent site-directed spin labeling (SDSL) studies identified specific parts of arrestin-1 engaged by different functional forms of rhodopsin and provided direct evidence of binding-induced conformational changes (11,12). A conformational change in the so-called finger loop (Fig.…”

“…Interestingly, it contains four conformationally distinct monomers (chains A-D) showing the natural plasticity of the arrestin-1 molecule. For example, the "finger loop" (residues 67-79), which is highly conserved in the arrestin family (29) and has been identified as a key receptor-binding element in two subtypes (11,30), has two distinct conformations in the crystal structure, bent (chain D) and extended (chain A) (Fig. 3A).…”