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 (P-Rh*). 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 P-Rh* 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.

Fig. 1.

Arrestin-1 binding to dark-state P-Rh. (A) Superimposed ¹H,¹⁵N-TROSY spectra of 30 μM ²H,¹⁵N-labeled arrestin-1 titrated by P-Rh in 4.2% anionic bicelles, 25 mM Bis●Tris, 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT pH 6.5 at 308 K. The bicelles contained DMPC:DMPG = 4:1 (mol:mol), q = 0.3, where q is the mol:mol ratio of the detergent-like D7PC to DMPC+DMPG. All samples in this work were examined in the same buffer and bicelles and at the same temperature. The molar ratio of arrestin-1 to P-Rh was varied: 1:0 (black), 1:1 (green), 1:2 (red), and 1:4 (cyan). (B) Mapping of P-Rh–induced site-specific NMR chemical-shift changes onto the 3D structure of arrestin-1 (PDB ID 1CF1). Red sites are those for which the backbone amide chemical shifts changed by 0.010 ppm or more. Light blue sites are those for which peaks shifted by less than 0.010 ppm, but underwent significant line-broadening in response to P-Rh. Resonances for dark blue sites neither shifted significantly nor broadened. Because the crystal structure does not resolve residues 1–9, 363–373, and 394–404, these segments were modeled into the depicted structure using X-PLOR-NIH (http://nmr.cit.nih.gov/xplor-nih).

Fig. 2.

Binding of arrestin to P-Rh*. (A) (red) TROSY spectrum of 30 μM ²H,¹⁵N-labeled arrestin-1 complexed with P-Rh* in bicelles. The spectrum of 30-μM free arrestin-1 (black) shows substantial chemical shift differences for the residues that remain observable. (B) Spectrum of arrestin-1 bound to P-Rh* from A, with labeling of the assigned peaks. (C) Determination of K_D for binding of arrestin-1 with P-Rh* (Left) or with P-opsin (Right).

Fig. 3.

800 MHz ¹H,¹³C-Me-TROSY of arrestin-1 binding to P-Rh*. (Upper) Isoleucine region of arrestin-1 spectrum. (Lower) Leucine and valine region of spectrum. All three samples contained 4% bicelles at 308 K and pH 6.5. Arrestin-1 was perdeuterated except for the protonated methyl groups of isoleucine, leucine, and valine, which were also ¹³C-labeled. The sample represented by the black spectrum contained 90 μM unlabeled P-Rh*, but no arrestin-1 (only the set of natural abundance ¹H,¹³C-methyl peaks from the bicelles were observed). The sample represented by the red spectrum contained 30 μM arrestin-1 but no P-Rh*. The light-blue spectrum corresponds to a sample containing both 30 μM labeled arrestin-1 and 90 μM unlabeled P-Rh*.