July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Divergent conformations of the arrestin-rhodopsin complex in solution
Author Affiliations & Notes
  • Sergey A Vishnivetskiy
    Pharmacology, Vanderbilt University, Nashville, Tennessee, United States
  • Matthias Elgeti
    Chemistry and Biochemistry, Jules Stein Eye Institute, University of California, Los Angeles, California, United States
  • Ned Van Eps
    Biochemistry, University of Toronto, Toronto, Ontario, Canada
  • Nicole A Perry
    Pharmacology, Vanderbilt University, Nashville, Tennessee, United States
  • Wayne Hubbell
    Chemistry and Biochemistry, Jules Stein Eye Institute, University of California, Los Angeles, California, United States
  • Vsevolod V Gurevich
    Pharmacology, Vanderbilt University, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Sergey Vishnivetskiy, None; Matthias Elgeti, None; Ned Van Eps, None; Nicole Perry, None; Wayne Hubbell, None; Vsevolod Gurevich, None
  • Footnotes
    Support  RO1 EY011500 (VVG)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 559. doi:
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      Sergey A Vishnivetskiy, Matthias Elgeti, Ned Van Eps, Nicole A Perry, Wayne Hubbell, Vsevolod V Gurevich; Divergent conformations of the arrestin-rhodopsin complex in solution. Invest. Ophthalmol. Vis. Sci. 2019;60(9):559.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The goal of this study is to directly examine the conformational heterogeneity of the arrestin-rhodopsin complex.

Methods : High-resolution distance measurements using site-directed spin labeling (SDSL) and double electron-electron resonance (DEER) were performed between selected sites in bovine arrestin-1 (60, 89, 131, 140, and 240) and monomeric bovine rhodopsin (74, 225, 235, and 308) in nanodiscs. Single-cysteine mutants of enhanced phosphorylation-independent arrestin-1 and rhodopsin were expressed and purified. Rhodopsin was reconstituted into lipid nanodiscs. Both proteins were chemically modified with the R1 spin label side chain, rhodopsin was light-activated and allowed to bind arrestin-1. Thereafter intermolecular distances distributions between both proteins were determined using DEER spectroscopy.

Results : For each intermolecular distance pair we detected multiple distances, some of which match the crystal structure of the arrestin-receptor complex. For example, we observed four distances (22 Å, 28 Å, 37 Å, and 43 Å) between arrestin-60R1 and rhodopsin-74R1, where only the distance of 28 Å matches the crystal structure. Similar results we acquired for other pairs of spin label positions in arrestin and rhodopsin. The presence of multiple distinct distances detected by DEER in each case suggests that different shapes of this complex exist, which have not been captured by crystallization. The DEER data for the arrestin-rhodopsin complex are in agreement with the flexibility of rhodopsin-bound arrestin-1 earlier detected using biophysical methods EPR and NMR that reveal protein dynamics. Collectively, our data suggest that the orientation of the two proteins in the complex relative to each other can be different and that active receptor-bound arrestin can assume several distinct conformations.

Conclusions : The arrestin-rhodopsin complex is flexible, existing in multiple conformations. As rhodopsin is a prototypical class A G protein-coupled receptor, these data reveal the structural basis of functional diversity of the arrestin-receptor complexes.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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