Abstract
Arrestin-1 selectively binds active phosphorylated rhodopsin (P-Rh*), demonstrating much lower affinity for inactive phosphorylated (P-Rh) and unphosphorylated active (Rh*) forms. Receptor interaction induces significant conformational changes in arrestin-1, which include large movement of the previously neglected 139-loop in the center of the receptor binding surface, away from the incoming receptor. To elucidate the functional role of this loop, in mouse arrestin-1 we introduced deletions of variable lengths and made several substitutions of Lys-142 in it and Asp-72 in the adjacent loop. Several mutants with perturbations in the 139-loop demonstrate increased binding to P-Rh*, dark P-Rh, Rh*, and phospho-opsin. Enhanced binding of arrestin-1 mutants to non-preferred forms of rhodopsin correlates with decreased thermal stability. The 139-loop perturbations increase P-Rh* binding of arrestin-1 at low temperatures and further change its binding profile on the background of 3A mutant, where the C-tail is detached from the body of the molecule by triple alanine substitution. Thus, the 139-loop stabilizes basal conformation of arrestin-1 and acts as a brake, preventing its binding to non-preferred forms of rhodopsin. Conservation of this loop in other subtypes suggests that it has the same function in all members of the arrestin family.
Highlights
In our recent study of the conformation of the receptor-bound arrestin using intramolecular distance measurements in free and P-Rh*-associated states, we found that the 139-loop undergoes large scale movement toward the N-domain and to the side of the molecule, apparently getting out of the way of the receptor, which engages the adjacent finger loop [25]
Two short deletions in the 139-loop of bovine arrestin-1 facilitate its binding to non-preferred forms of rhodopsin, Rh* and dark P-Rh, and one of these deletions induces the release of the arrestin-1 C-tail [25], which is a hallmark of its “activation” by bound receptor [38, 40]
Arrestins have several positively charged phosphate binding residues [28, 31, 37, 61] and multiple other exposed side chains that recognize the active receptor conformation [33, 39, 41]. Both parts of the interaction interface contribute to binding energy and at the same time serve as sensors
Summary
Conclusion: The central 139-loop supports basal arrestin-1 conformation and reduces its binding to non-preferred forms of rhodopsin. Enhanced binding of arrestin-1 mutants to non-preferred forms of rhodopsin correlates with decreased thermal stability. The 139loop stabilizes basal conformation of arrestin-1 and acts as a brake, preventing its binding to non-preferred forms of rhodopsin. Conservation of this loop in other subtypes suggests that it has the same function in all members of the arrestin family. Even mild perturbations of this loop are surprisingly detrimental for the thermal stability of the protein and promote arrestin-1 binding to non-preferred functional forms of rhodopsin at the same time. Remarkable structural conservation of this loop in all members of the arrestin family (26 –29) suggests that it likely enhances stability and selectivity of other arrestin subtypes
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