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Sergey A. Vishnivetskiy, Miyeon Kim, Ned Van Eps, Maria C. Palazzo, Britney N. Lizama, Wayne L. Hubbell, Vsevolod V. Gurevich; Central 139-loop Enhances Stability And Selectivity For P-rh* Of Arrestin-1. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4131.
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To determine the function of 139 loop in arrestin-1.
Site-directed spin labeling and distance measurements by Double Electron-Electron Resonance (DEER) were used to monitor the movement of 139-loop upon arrestin-1 binding to light-activated phosphorhodopsin (P-Rh*). Direct binding assay with 139-loop deletion mutants was used to determine its role in arrestin-1 selectivity and stability.
The central crest on the rhodopsin-binding side of arrestin-1 contains two loops: the finger loop, involved in P-Rh* binding, and 139-loop. The mobility of the spin label in position 139 is reduced upon arrestin-1 pre-docking to dark P-Rh, but reverts to the initial high level upon rhodopsin activation to P-Rh*, suggesting that it moves away from the receptor in high-affinity complex. DEER distance measurements between the spin label at 139 and in eight different positions in N- and C-domain show that upon P-Rh* binding 139-loop moves by >10Å towards the N-domain and to the side of the molecule. This shift removes 139-loop from the receptor-binding interface, yet 139-loop is conserved in all arrestins, suggesting that it plays an important biological role. We introduced four deletions of 139-loop of variable length, and found that two of these reduced arrestin-1 selectivity for P-Rh* by increasing binding to dark P-Rh and Rh*. The same two deletions dramatically decreased arrestin-1 stability, whereas the other two reduced selectivity and stability only modestly. Homologous deletions in bovine and mouse arrestin-1 yielded virtually identical effects on selectivity and stability. Thus, 139-loop deletion mutants follow the same pattern as previously described pre-activated forms of arrestin-1: protein stability inversely correlates with its ability to bind dark P-Rh and Rh*.
In arrestin-1, 139-loop has two related functions: 1) it stabilizes the basal conformation; 2) it serves as a brake, minimizing binding to non-preferred forms of rhodopsin, thereby ensuring its specificity for P-Rh*.
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