Monitoring Interactions Between S100B and the Dopamine D2 Receptor Using NMR Spectroscopy

  • Yuning Wang
  • Roya Tadayon
  • Gary S. ShawEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)


S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and interacts with a wide range of proteins to modulate their functions. The dopamine D2 receptor is one potential S100B binding partner that may play a key role in neurological processing. In this chapter, we describe the use of NMR spectroscopy to examine the interaction between calcium-bound S100B and the third intracellular loop (IC3) from the dopamine D2 receptor. We provide details that allow the strength of the interaction (Kd) between the two proteins to be determined and the IC3 site of interaction on the structure of S100B to be identified. Both these characteristics can be identified from a single series of nondestructive experiments.

Key words

S100B Dopamine receptor Protein interactions Binding site Protein structure 



This research was supported by a research grant (MOP 93520) from the Canadian Institutes of Health Research (GSS). We thank Brian Dempsey for the helpful discussions. Yuning Wang and Roya Tadayon contributed equally to this work.


  1. 1.
    Luker KE, Piwnica-Worms D (2004) Optimizing luciferase protein fragment complementation for bioluminescent imaging of protein-protein interactions in live cells and animals. Methods Enzymol 385:349–360. CrossRefPubMedGoogle Scholar
  2. 2.
    Nohe A, Petersen NO (2004) Analyzing protein-protein interactions in cell membranes. BioEssays 26:196–203. CrossRefPubMedGoogle Scholar
  3. 3.
    Donato R, Sorci G, Riuzzi F et al (2009) S100B’s double life: intracellular regulator and extracellular signal. Biochim Biophys Acta 1793:1008–1022. CrossRefPubMedGoogle Scholar
  4. 4.
    Liu J, Wang H, Zhang L et al (2011) S100B transgenic mice develop features of Parkinson’s disease. Arch Med Res 42:1–7. CrossRefPubMedGoogle Scholar
  5. 5.
    Leclerc E, Sturchler E, Vetter SW (2010) The S100B/RAGE axis in Alzheimer’s disease. Cardiovasc Psychiatry Neurol 2010:539581. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Rezvanpour A, Shaw GS (2009) Unique S100 target protein interactions. Gen Physiol Biophys 28:F39–F46CrossRefGoogle Scholar
  7. 7.
    Santamaria-Kisiel L, Rintala-Dempsey AC, Shaw GS (2006) Calcium-dependent and -independent interactions of the S100 protein family. Biochem J 396:201–214. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Heizmann CW, Fritz G, Schäfer BW (2002) S100 proteins: structure, functions and pathology. Front Biosci 7:d1356–d1368. CrossRefPubMedGoogle Scholar
  9. 9.
    Fritz G, Heizmann CW (2006) 3D Structures of the calcium and zinc binding S100 proteins. In: Handbook of metalloproteins. John Wiley & Sons, Ltd, Hoboken, NJ. CrossRefGoogle Scholar
  10. 10.
    Ostendorp T, Leclerc E, Galichet A et al (2007) Structural and functional insights into RAGE activation by multimeric S100B. EMBO J 26:3868–3878. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    McClintock KA, Shaw GS (2003) A novel S100 target conformation is revealed by the solution structure of the Ca2+-S100B-TRTK-12 complex. J Biol Chem 278:6251–6257. CrossRefPubMedGoogle Scholar
  12. 12.
    Inman KG, Yang R, Rustandi RR et al (2002) Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12. J Mol Biol 324:1003–1014. CrossRefPubMedGoogle Scholar
  13. 13.
    Gógl G, Alexa A, Kiss B et al (2016) Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein. J Biol Chem 291:11–27. CrossRefPubMedGoogle Scholar
  14. 14.
    Rustandi RR, Drohat AC, Baldisseri DM et al (1998) The Ca2+-dependent interaction of S100B(ββ) with a peptide derived from p53. Biochemistry 37:1951–1960. CrossRefPubMedGoogle Scholar
  15. 15.
    Rustandi RR, Baldisseri DM, Weber DJ (2000) Structure of the negative regulatory domain of p53 bound to S100B(ββ). Nat Struct Biol 7:570–574. CrossRefPubMedGoogle Scholar
  16. 16.
    Donato R, Cannon BR, Sorci G et al (2013) Functions of S100 proteins. Curr Mol Med 13:24–57. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Liu Y, Buck DC, Neve KA (2008) Novel interaction of the dopamine D2 receptor and the Ca2+ binding protein S100B: role in D2 receptor function. Mol Pharmacol 74:371–378. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Moritz AE, Benjamin Free R, Sibley DR (2018) Advances and challenges in the search for D2 and D3 dopamine receptor-selective compounds. Cell Signal 41:75–81. CrossRefPubMedGoogle Scholar
  19. 19.
    Urs NM, Peterson SM, Caron MG (2017) New concepts in dopamine D2 receptor biased signaling and implications for Schizophrenia therapy. Biol Psychiatry 81:78–85. CrossRefPubMedGoogle Scholar
  20. 20.
    Katzung BG (2001) Introduction to autonomic pharmacology. In: Basic and clinical pharmacology, 8th edn. The McGraw Hill Companies, Inc, New York, NYGoogle Scholar
  21. 21.
    Stanwood GD (2008) Protein-protein interactions and dopamine D2 receptor signaling: a calcium connection. Mol Pharmacol 74:317–319. CrossRefPubMedGoogle Scholar
  22. 22.
    Senogles SE, Heimert TL, Odife ER, Quasney MW (2004) A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity. J Biol Chem 279:1601–1606. CrossRefPubMedGoogle Scholar
  23. 23.
    Dempsey BR, Shaw GS (2011) Identification of calcium-independent and calcium-enhanced binding between S100B and the dopamine D2 receptor. Biochemistry 50:9056–9065. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of protein-protein interactions in vivo. Plant Physiol 171:727–758. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    O’Connell MR, Gamsjaeger R, Mackay JP (2009) The structural analysis of protein-protein interactions by NMR spectroscopy. Proteomics 9:5224–5232. CrossRefPubMedGoogle Scholar
  26. 26.
    Smith SP, Barber KR, Dunn SD, Shaw GS (1996) Structural influence of cation binding to recombinant human brain S100b: Evidence for calcium-induced exposure of a hydrophobic surface. Biochemistry 35:8805–8814. CrossRefPubMedGoogle Scholar
  27. 27.
    Smith SP, Shaw GS (1997) Assignment and secondary structure of calcium-bound human S100B. J Biomol NMR 10:77–88CrossRefGoogle Scholar
  28. 28.
    Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293. CrossRefPubMedGoogle Scholar
  29. 29.
    Johnson BA, Blevins RA (1994) NMR View: A computer program for the visualization and analysis of NMR data. J Biomol NMR 4:603–614. CrossRefPubMedGoogle Scholar
  30. 30.
    Baryshnikova OK, Williams TC, Sykes BD (2008) Internal pH indicators for biomolecular NMR. J Biomol NMR 41:5–7. CrossRefPubMedGoogle Scholar
  31. 31.
    Rintala-Dempsey AC, Santamaria-Kisiel L, Liao Y et al (2006) Insights into S100 target specificity examined by a new interaction between S100A11 and annexin A2. Biochemistry 45:14695–14705. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BiochemistryThe University of Western OntarioLondonCanada

Personalised recommendations