The single nucleotide ß-arrestin2 variant, A248T, resembles dynamical properties of activated arrestin

ß-arrestins are responsible for termination of G protein-coupled receptor GPCR -mediated signaling. Association of single nucleotide variants with onset of crucial diseases has made this protein family hot targets in the field of GPCR-mediated pharmacology. However, impact of these mutations on function of these variants has remained elusive. In this study, structural and dynamical properties of one of ß-arrestin2 arrestin 3 variants, A248T, which has been identified in some cancer tissue samples, were investigated via molecular dynamics simulations. The results showed that the variant underwent structural rearrangements which are seen in crystal structures of active arrestin. Specifically, the "short helix" unravels and the "gate loop" swings forward as seen in crystal structures of receptor-bound and GPCR phosphopeptide-bound arrestin. Moreover, the "finger loop" samples upward position in the variant. Importantly, these regions harbor crucial residues that are involved in receptor binding interfaces. Cumulatively, these local structural rearrangements help the variant adopt active-like domain angle without perturbing the "polar core". Considering that phosphorylationofthereceptorisrequiredforactivationofarrestin, A248Tmightserveasamodelsystemtounderstand phosphorylation-independent activation mechanism, thus enabling modulation of function of arrestin variants which are activated independent of receptor phosphorylation as seen in cancer.

Keywords:

Arrestin, G protein-coupled receptor, phosphorylation-independent activation, cancer, single nucleotide polymorphism molecular dynamics simulation,

PDF

___

1. Freedman NJ, Lefkowitz RJ. Desensitization of G protein-coupled receptors. Recent Progress in Hormone Research 1996; 51: 319-351. doi: 10.1146/annurev.neuro.27.070203.144206
2. Hausdorff WP, Caron MG, Lefkowitz RJ. Turning off the signal: desensitization of beta-adrenergic receptor function. Federation of American Societies For Experimental Biology Journal 1990; 4 (11): 2881-2889. doi: 10.1096/fasebj.4.11.2165947
3. Goodman OB, Krupnick JG, Santini F, Gurevich VV, Penn RB et al. Role of arrestins in G-protein-coupled receptor endocytosis. Advances in Pharmacology 1998; 42: 429-433. doi: 10.1016/s1054-3589(08)60780-2
4. Gimenez LE, Kook S, Vishnivetskiy SA, Ahmed MR, Gurevich EV et al. Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors. Journal of Biological Chemistry 2012; 287: 9028-9040. doi: 10.1074/jbc.M111.311803
5. Kovoor A, Celver J, Abdryashitov RI, Chavkin C, Gurevich VV. Targeted construction of phosphorylationindependent beta-arrestin mutants with constitutive activity in cells. Journal of Biological Chemistry 1999; 274: 6831-6834. doi: 10.1074/jbc.274.11.6831
6. Gurevich VV, Dion SB, Onorato JJ, Ptasienski J, Kim CM et al. Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild type and mutant arrestins with rhodopsin, beta 2-adrenergic, and m2 muscarinic cholinergic receptors. Journal of Biological Chemistry 1995; 270: 720-731. doi: 10.1074/jbc.270.2.720
7. Shukla AK, Westfield GH, Xiao K, Reis RI, Huang LY et al. Visualization of arrestin recruitment by a G-proteincoupled receptor. Nature 2014; 512: 218-222. doi: 10.1038/nature13430
8. Kim Y, Hofmann K, Ernst O, Scheerer P, Choe H et al. Crystal structure of pre-activated arrestin p44. Nature 2013; 497: 142-146. doi: 10.1038/nature12133
9. Vishnivetskiy SA, Schubert C, Climaco GC, Gurevich YV, Velez MG et al. An additional phosphate-binding element in arrestin molecule. Implications for the mechanism of arrestin activation. Journal of Biological Chemistry 2000; 275: 41049-41057. doi: 10.1074/jbc.M007159200
10. Granzin J, Stadler A, Cousin A, Schlesinger R, Batra-Safferling R. Structural evidence for the role of polar core residue Arg175 in arrestin activation. Scientific Reports 2015; 5: 15808. doi: 10.1038/srep15808
11. Latorraca NR, Wang JK, Bauer B, Townshend RJL, Hollingsworth SA et al. Molecular mechanism of GPCRmediated arrestin activation. Nature 2018; 557: 452-456. doi: 10.1038/s41586-018-0077-3
12. Shukla AK, Manglik A, Kruse AC, Xiao K, Reis RI et.al. Structure of active beta-arrestin1 bound to a G proteincoupled receptor phosphopeptide. Nature 2013; 497: 137-141. doi: 10.1038/nature12120
13. . Zhou XE, He Y, de Waal PW, Gao X, Kang Y et al. Identification of phosphorylation codes for arrestin recruitment by G protein-coupled receptors. Cell 2017; 170: 457-469. doi: 10.1016/j.cell.2017.07.002
14. Sensoy O, Moreira IS, Morra G. Understanding the differential selectivity of arrestins toward the phosphorylation state of the receptor. ACS Chemical Neuroscience 2016; 7 (9): 1212-1224. doi: 10.1021/acschemneuro.6b00073
15. Zheng C, Tholen J, Gurevich VV. Critical role of the finger loop in arrestin binding to the receptors. Plos One 2019; 14 (3): e0213792. doi: 10.1371/journal.pone.0213792
16. Shihab HA, Gough J, Cooper DN, Stenson PD, Barker GLA et al. Predicting the functional, molecular, and phenotypic consequences of amino acid substitutions using hidden markov models. Human Mutation 2013; 34 (1): 57–65. doi: 10.1002/humu.22225
17. Hirsch JA, Schubert C, Gurevich VV, Sigler PB. The 2.8 A crystal structure of visual arrestin: a model for arrestin’s regulation. Cell 1999; 97: 257-269. doi: 10.1016/s0092-8674(00)80735-7
18. Zhan X, Gimenez LE, Gurevich VV, Spiller BW. Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual subtypes. Journal of Molecular Biology 2011; 406: 467-478. doi: 10.1016/j.jmb.2010
19. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006; 22: 195-201. doi: 10.1093/bioinformatics/bti770
20. DeLano WL. Pymol: An open-source molecular graphics tool. CCP4 Newsletter On Protein Crystallography 2002; 40: 82-92.
21. Bas DC, Rogers DM, Jensen JH. Very fast prediction and rationalization of pKa values for protein–ligand complexes. Proteins: Structure, Function, Bioinformatics 2008; 73: 765-783. doi: 10.1002/prot.22102
22. Olsson MHM, Sondergaard CR, Rostkowski M, Jensen JH. PROPKA3: Consistent treatment of internal and surface residues in empirical pKa predictions. Journal of Chemical Theory and Computation 2011; 7: 525-537. doi: 10.1021/ct100578z
23. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC et al. Gromacs: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 2015; 1-2: 19-25. doi: 10.1016/j.softx.2015.06.001
24. Huang J, MacKerell AD Jr. Charmm36 all-atom additive protein force field: validation based on comparison to NMR data. Journal of Computational Chemistry 2013; 34 (25): 2135-45. doi: 10.1002/jcc.23354
25. Mark P, Nilsson L. Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. Journal of Physical Chemistry A 2001; 105: 9954-9960. doi: 10.1021/jp003020w
26. Berendsen HJC,Postma JPM,Gunsteren VF,DiNola A,Haak JR. Molecular dynamics with coupling to an external bath. Journal of Chemical Physics 1984; 81: 3684. doi: 10.1063/1.448118
27. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H et al. A smooth particle mesh Ewald method. Journal of Chemical Physics 1995; 103: 8577-8593. doi: 10.1063/1.470117
28. Ryckaert JP, Ciccotti G, Berendsen H. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. Journal of Computational Physics 1977; 23: 327-341. doi: 10.1016/0021-9991(77)90098-5
29. Heinig M, Frishman D. STRIDE: a web server for secondary structure assignment from known atomic coordinates of proteins. Nucleic Acids Research 2004; 32(Web Server issue): W500–W502. doi: 10.1093/nar/gkh429
30. Humphrey W, Dalke A, Schulten K. VMD: Visual molecular dynamics. Journal of Molecular Graphics 1996; 14: 33-38. doi: 10.1016/0263-7855(96)00018-5
31. Hess B, Kutzner C, van der Spoel D, Lindahl E. Gromacs 4: Algorithms for highly efficient, load-balanced, and scalable molecular simulation. Journal of Chemical Theory and Computation 2008; 4: 435-447. doi: 10.1021/ct700301q
32. Sommer M, Smith WC, Farrens DL. Dynamics of arrestin-rhodopsin interactions: Arrestin and retinal release are directly linked events. Journal of Biological Chemistry 2005; 280: 6861-6871. doi: 10.1074/jbc.M411341200
33. Sommer M, Farrens DL, McDowell JH, Weber LA, Smith WC. Dynamics of arrestin-rhodopsin interactions: Loop movement is involved in arrestin activation and receptor binding. Journal of Biological Chemistry 2007; 282: 25560-25568. doi: 10.1074/jbc.M702155200
34. Kim M, Vishnivetskiyc SA, Eps NV, Alexander NS, Cleghorn WM et al. Conformation of receptor-bound visual arrestin. Proceedings of National Science Academy 2012; 109: 18407-18412. doi: 10.1073/pnas.1216304109
35. Zhou XE, Gao X, Barty A, Kang Y, He Y et al. X-ray laser diffraction for structure determination of the rhodopsin-arrestin complex. Scientific Data 2016; 3: 160021. doi: 10.1038/sdata.2016.21