NMR Structure of the “Finger” Loop of Rod Arrestin Induced by Meta-II Rhodopsin Binding

Abstract

An unstructured “finger” loop connects β-strands V and VI in x-ray crystal structures of arrestin. Several recent studies identified this region as a possible major interaction site for specific binding of arrestin to photo-activated and/or phosphorylated meta-II rhodopsin. We studied binding of a peptide representing the amino acid sequence of the loop around residue 75 of visual arrestin to light-activated bovine rhodopsin. At low millimolar concentrations the peptide enhances formation of the photointermediate meta-II rhodopsin after receptor activation. The extra meta-II assay reveals competition of the peptide with transducin and arrestin for binding to activated (Rh*) and activated phosphorylated rhodopsin (P-Rh*), respectively. The high resolution structure of the meta-II rhodopsin-bound arrestin peptide was derived from the difference of two-dimensional liquid state NMR spectra recorded in the presence of either dark-adapted or light-activated rhodopsin-rich disc membranes. The peptide remains unstructured upon addition of dark-adapted rhodopsin. However, receptor activation causes dramatic changes in 2D NOESY spectra of the peptide. Specifically, the ligand is in rapid exchange between a free unstructured and a conformationally well defined meta-II-bound form. Efficient cross-relaxation of proton spins in the bound state causes strong NOESY cross-peaks reflecting the bound peptide structure. The specific pattern of binding-induced NOESY peaks indicates a helical conformation of the bound peptide. Restrained molecular dynamics calculations confirmed an α-helical peptide structure. A structural model of the interface between meta-II rhodopsin and the arrestin loop region is proposed based on molecular docking.