Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin

Abstract

Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (PRh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Although arrestin-1 affinity for Rh* was seen to be low (K D > 150 μM), its affinity for P-Rh (K D ∼80 μM) was comparable to the concentration of active monomeric arrestin-1 in the outer segment, suggesting that P-Rh generated by high-gain phosphorylation is occupied by arrestin-1 under physiological conditions and will not signal upon photo-activation. Arrestin-1 was seen to bind P-Rh* and P-opsin with fairly high affinity (K D of ∼50 and 800 nM, respectively), implying that arrestin-1 dissociation is triggered only upon P-opsin regeneration with 11-cis-retinal, precluding noise generated by opsin activity. Based on their observed affinity for arrestin-1, P-opsin and inactive P-Rh very likely affect the physiological monomer-dimer-tetramer equilibrium of arrestin-1, and should therefore be taken into account when modeling photoreceptor function. The data also suggested that complex formation with either PRh* or P-opsin results in a global transition in the conformation of arrestin-1, possibly to a dynamic molten globule-like structure. We hypothesize that this transition contributes to the mechanism that triggers preferential interactions of several signaling proteins with receptor-activated arrestins.G protein-coupled receptors | nuclear magnetic resonance | TROSY | bicelles

Functional map of arrestin-1 at single amino acid resolution

“…Such conformational changes are in agreement with the structure of the β2-adrenergic receptor-G protein complex (6) which, in comparison with metarhodopsin-II (4,5) and an active nanobody stabilized β2-adrenergic receptor state (44), showed a large outward movement of TM6 upon G protein binding. In addition to rearrangements in the receptor, solution NMR suggested a global transition of arrestin-1 upon binding to rhodopsin, due to the adaptation of a dynamic molten globule-like structure (20). Conformational changes in both arrestin and rhodopsin upon complex formation may thus explain the large distance between the major interaction surfaces revealed by our scanning mutagenesis.…”

“…Release of the C tail is one of the major conformational changes occurring during arrestin activation as suggested by the sequential multisite binding model (17) and confirmed by EPR (24) and NMR (20) spectroscopic studies. The recent crystal structure of p44 arrestin-1, lacking the arrestin C tail, revealed marked conformational changes compared with a previous structure of p44 arrestin-1 (25), including a 20°rotation between the C and N domains (9) as had previously been suggested based on arrestin truncations (26) and molecular modeling (27).…”

“…1B). Importantly the halfmaximal inhibitory concentration (IC 50 ) for NaCl determined with this assay is largely independent from variations in the arrestin-1 expression level, as we chose a rhodopsin concentration of 1.25 μM, far in excess of the 5-50 nM apparent binding affinity of arrestin-1 (19,20). As a further precaution we expressed wild-type arrestin in parallel with each set of mutants.…”

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

“…In the basal state, the C terminus folds back toward the N-domain and forms a highly conserved tripartite interaction with the N-domain, involving ␤-strand I and ␣-helix I (4,21,25,26). Upon binding to its cognate GPCR rhodopsin, several conformational changes in arrestin-1 have been proposed and confirmed by studies using NMR, fluorescence quenching, and site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy techniques (25,(27)(28)(29)(30)(31)(32)(33)). An EPR study using double electron electron resonance (DEER) combined with RosettaEPR modeling provided a global picture of the phosphorylated activated rho-dopsin (PR*)-induced conformational changes in arrestin-1, which involve the release of the C-tail, the movement of the finger loop, the movement of a loop containing residue 139, and smaller changes in distal loops containing residues 157 and 344 at the tips of the N-and C-domains (31).…”

“…Interestingly, three distinct distance populations coexist between spin labels at 13 and 392 in the receptor-bound conformation, which suggests that the C-tail of arrestin-3 probably adopts several specific conformations, in contrast to arrestin-2 with a single distance (Fig. 3) and to the completely flexible and free C-tail in receptor-bound arrestin-1 (29,31,33). Conformational changes induced by PR* binding in the finger loop were tested using 68/168 arrestin-3.…”