The Structure of the Polar Core Mutant R175E and Its Functional Implications

  • Renu Batra-SafferlingEmail author
  • Joachim Granzin


Mutation of arginine 175 to glutamic acid (R175E), a central residue in the polar core and previously predicted as the ‘phosphosensor’, leads to a constitutively active arrestin that is able to terminate phototransduction by binding to non-phosphorylated, light-activated rhodopsin . Crystal structure of a R175E mutant arrestin at 2.7 Å resolution reveals significant differences compared to the basal state reported in full-length arrestin structures. Most striking differences are disruption of hydrogen bond network in the polar core , and three-element interaction (between β-strand I, α-helix I, and the C-tail), including disordering of several residues in the receptor-binding finger loop and the C-terminus (residues 361–404). Additionally, R175E structure shows a 7.5° rotation of the amino and carboxy-terminal domains relative to each other. Comparison of the crystal structures of basal arrestin and R175E mutant provides insights into the mechanism of arrestin activation, where the latter likely represents an intermediate activation state prior to formation of the high-affinity complex with the G protein-coupled receptor.


Arrestin Phosphorylation-independent Photoreceptor Polar core Rhodopsin 


  1. Celver J, Vishnivetskiy SA, Chavkin C, Gurevich VV (2002) Conservation of the phosphate-sensitive elements in the arrestin family of proteins. J Biol Chem 277:9043–9048CrossRefPubMedGoogle Scholar
  2. Feuerstein SE, Pulvermuller A, Hartmann R, Granzin J, Stoldt M, Henklein P, Ernst OP, Heck M, Willbold D, Koenig BW (2009) Helix formation in arrestin accompanies recognition of photoactivated Rhodopsin. Biochem US 48:10733–10742CrossRefGoogle Scholar
  3. Granzin J, Wilden U, Choe HW, Labahn J, Krafft B, Buldt G (1998) X-ray crystal structure of arrestin from bovine rod outer segments. Nature 391:918–921CrossRefPubMedGoogle Scholar
  4. Granzin J, Cousin A, Weirauch M, Schlesinger R, Buldt G, Batra-Safferling R (2012) Crystal structure of p44, a constitutively active splice variant of visual arrestin. J Mol Biol 416:611–618CrossRefPubMedGoogle Scholar
  5. Granzin J, Stadler A, Cousin A, Schlesinger R, Batra-Safferling R (2015) Structural evidence for the role of polar core residue Arg175 in arrestin activation. Sci Rep 5:15808CrossRefPubMedPubMedCentralGoogle Scholar
  6. Gray-Keller MP, Detwiler PB, Benovic JL, Gurevich VV (1997) Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion. Biochem US 36:7058–7063CrossRefGoogle Scholar
  7. Gurevich VV, Benovic JL (1993) Visual arrestin interaction with rhodopsin—sequential multisite binding ensures strict selectivity toward light-activated phosphorylated rhodopsin. J Biol Chem 268:11628–11638PubMedGoogle Scholar
  8. Gurevich VV, Benovic JL (1995) Visual arrestin binding to rhodopsin—diverse functional roles of positively charged residues within the phosphorylation-recognition region of arrestin. J Biol Chem 270:6010–6016CrossRefPubMedGoogle Scholar
  9. Gurevich VV, Benovic JL (1997) Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state. Mol Pharmacol 51:161–169PubMedGoogle Scholar
  10. Gurevich VV, Gurevich EV (2004) The molecular acrobatics of arrestin activation. Trends Pharmacol Sci 25:105–111CrossRefPubMedGoogle Scholar
  11. Gurevich VV, Hanson SM, Song XF, Vishnivetskiy SA, Gurevich EV (2011) The functional cycle of visual arrestins in photoreceptor cells. Prog Retin Eye Res 30:405–430CrossRefPubMedPubMedCentralGoogle Scholar
  12. Gurevich VV, Song X, Vishnivetskiy SA, Gurevich EV (2014) Enhanced phosphorylation-independent arrestins and gene therapy. Handb Exp Pharmacol 219:133–152CrossRefPubMedPubMedCentralGoogle Scholar
  13. Han M, Gurevich VV, Vishnivetskiy SA, Sigler PB, Schubert C (2001) Crystal structure of beta-arrestin at 1.9 angstrom: Possible mechanism of receptor binding and membrane translocation. Structure 9:869–880CrossRefPubMedGoogle Scholar
  14. Hanson SM, Gurevich VV (2006) The differential engagement of arrestin surface charges by the various functional forms of the receptor. J Biol Chem 281:3458–3462CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hanson SM, Francis DJ, Vishnivetskiy SA, Kolobova EA, Hubbell WL, Klug CS, Gurevich VV (2006) Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin. Proc Natl Acad Sci USA 103:4900–4905CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hirsch JA, Schubert C, Gurevich VV, Sigler PB (1999) The 2.8 angstrom crystal structure of visual arrestin: a model for arrestin’s regulation. Cell 97:257–269CrossRefPubMedGoogle Scholar
  17. Kang Y, Zhou XE, Gao X, He Y, Liu W, Ishchenko A, Barty A, White TA, Yefanov O, Han GW, Xu Q, de Waal PW, Ke J, Tan MH, Zhang C, Moeller A, West GM, Pascal BD, Van Eps N, Caro LN, Vishnivetskiy SA, Lee RJ, Suino-Powell KM, Gu X, Pal K, Ma J, Zhi X, Boutet S, Williams GJ, Messerschmidt M, Gati C, Zatsepin NA, Wang D, James D, Basu S, Roy-Chowdhury S, Conrad CE, Coe J, Liu H, Lisova S, Kupitz C, Grotjohann I, Fromme R, Jiang Y, Tan M, Yang H, Li J, Wang M, Zheng Z, Li D, Howe N, Zhao Y, Standfuss J, Diederichs K, Dong Y, Potter CS, Carragher B, Caffrey M, Jiang H, Chapman HN, Spence JC, Fromme P, Weierstall U, Ernst OP, Katritch V, Gurevich VV, Griffin PR, Hubbell WL, Stevens RC, Cherezov V, Melcher K, Xu HE (2015) Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523:561–567CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kim M, Vishnivetskiy SA, Van Eps N, Alexander NS, Cleghorn WM, Zhan XZ, Hanson SM, Morizumi T, Ernst OP, Meiler J, Gurevich VV, Hubbell WL (2012) Conformation of receptor-bound visual arrestin. Proc Natl Acad Sci USA 109:18407–18412CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kim YJ, Hofmann KP, Ernst OP, Scheerer P, Choe HW, Sommer ME (2013) Crystal structure of pre-activated arrestin p44. Nature 497:142–146CrossRefPubMedGoogle Scholar
  20. Kovoor A, Celver J, Abdryashitov RI, Chavkin C, Gurevich VV (1999) Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells. J Biol Chem 274:6831–6834CrossRefPubMedGoogle Scholar
  21. Milano SK, Pace HC, Kim YM, Brenner C, Benovic JL (2002) Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis. Biochem US 41:3321–3328CrossRefGoogle Scholar
  22. Modzelewska A, Filipek S, Palczewski K, Park PS (2006) Arrestin interaction with rhodopsin: conceptual models. Cell Biochem Biophys 46:1–15CrossRefPubMedGoogle Scholar
  23. Ostermaier MK, Peterhans C, Jaussi R, Deupi X, Standfuss J (2014a) Functional map of arrestin-1 at single amino acid resolution. Proc Natl Acad Sci USA 111:1825–1830CrossRefPubMedPubMedCentralGoogle Scholar
  24. Ostermaier MK, Schertler GFX, Standfuss J (2014b) Molecular mechanism of phosphorylation-dependent arrestin activation. Curr Opin Struct Biol 29:143–151CrossRefPubMedGoogle Scholar
  25. Palczewski K, Pulvermuller A, Buczylko J, Hofmann KP (1991) Phosphorylated rhodopsin and heparin induce similar conformational-changes in arrestin. J Biol Chem 266:18649–18654PubMedGoogle Scholar
  26. Pan L, Gurevich EV, Gurevich VV (2003) The nature of the arrestin x receptor complex determines the ultimate fate of the internalized receptor. J Biol Chem 278:11623–11632CrossRefPubMedGoogle Scholar
  27. Shukla AK, Manglik A, Kruse AC, Xiao K, Reis RI, Tseng WC, Staus DP, Hilger D, Uysal S, Huang LY, Paduch M, Tripathi-Shukla P, Koide A, Koide S, Weis WI, Kossiakoff AA, Kobilka BK, Lefkowitz RJ (2013) Structure of active beta-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497:137–141CrossRefPubMedPubMedCentralGoogle Scholar
  28. Sutton RB, Vishnivetskiy SA, Robert J, Hanson SM, Raman D, Knox BE, Kono M, Navarro J, Gurevich VV (2005) Crystal structure of cone arrestin at 2.3A: evolution of receptor specificity. J Mol Biol 354:1069–1080CrossRefPubMedGoogle Scholar
  29. Vishnivetskiy SK, Paz CL, Schubert C, Hirsch JA, Sigler PB, Gurevich VV (1999) How does arrestin respond to the phosphorylated state of rhodopsin? J Biol Chem 274:11451–11454CrossRefPubMedGoogle Scholar
  30. Vishnivetskiy SA, Schubert C, Climaco GC, Gurevich YV, Velez MG, Gurevich VV (2000) An additional phosphate-binding element in arrestin molecule—implications for the mechanism of arrestin activation. J Biol Chem 275:41049–41057CrossRefPubMedGoogle Scholar
  31. Vishnivetskiy SA, Hirsch JA, Velez MG, Gurevich YV, Gurevich VV (2002) Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge. J Biol Chem 277:43961–43967CrossRefPubMedGoogle Scholar
  32. Vishnivetskiy SA, Baameur F, Findley KR, Gurevich VV (2013) Critical role of the central 139-loop in stability and binding selectivity of arrestin-1. J Biol Chem 288:11741–11750CrossRefPubMedPubMedCentralGoogle Scholar
  33. Wilden U, Hall SW, Kuhn H (1986) Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. Proc Natl Acad Sci USA 83:1174–1178CrossRefPubMedPubMedCentralGoogle Scholar
  34. Zhuang TD, Chen QY, Cho MK, Vishnivetskiy SA, Iverson TM, Gurevich VV, Sanders CR (2013) Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin. Proc Natl Acad Sci USA 110:942–947CrossRefPubMedGoogle Scholar
  35. Zhuo Y, Vishnivetskiy SA, Zhan XZ, Gurevich VV, Klug CS (2014) Identification of receptor binding-induced conformational changes in non-visual arrestins. J Biol Chem 289:20991–21002CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG 2017

Authors and Affiliations

  1. 1.Institute of Complex Systems (ICS-6), Structural BiochemistryForschungszentrum JülichJülichGermany

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