Skip to main content
SpringerLink
Log in

Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin

  • Protocol
Estrogen Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1366))

Abstract

The estrogen receptor α ligand-binding domain (ERα-LBD) binds the natural hormone 17β-estradiol (E2) to induce transcription and cell proliferation. This process occurs with the contribution of protein and peptide partners (also called coactivators) that can modulate the structure of ERα, and therefore its specificity of action. As with most transcription factors, ERα exhibits a high content of α helix, making it difficult to routinely run spectroscopic studies capable of deciphering the secondary structure of the different partners under binding conditions. Ca2+-calmodulin, a protein also highly structured in α-helix, is a key coactivator for ERα activity. Here, we show how circular dichroism can be used to study the interaction of ERα with Ca2+-calmodulin. Our approach allows the determination not only of the conformational changes induced upon complex formation but also the dissociation constant (Kd) of this interaction.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Schwabe JW, Chapman L, Finch JT, Rhodes D (1993) The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567–578

    Article  CAS  Google Scholar 

  2. Klinge C (2001) Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res 29:2905–2919

    Article  CAS  Google Scholar 

  3. Greenfield N, Vijayanathan V, Thomas TJ, Gallo MA, Thomas T (2006) Increase in the stability and helical content of estrogen receptor α in the presence of the estrogen response element: analysis by circular dichroism spectroscopy. Biochemistry 40:6646–6652

    Article  Google Scholar 

  4. Deegan BJ, Bhat V, Seldeen KL, McDonald CB, Farooq A (2011) Genetic variations within the ERE motif modulates plasticity and energetics of binding of DNA to the ERα nuclear receptor. Arch Biochem Biophys 507:262–270

    Article  CAS  Google Scholar 

  5. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL (1998) The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937

    Article  CAS  Google Scholar 

  6. Oostenbrinck BC, Pitera JW, van Lipzig MMH, Meerman JHN, van Gunsteren WF (2000) Simulations of the estrogen receptor ligand-binding domain: affinity of natural ligands and xenoestrogens. J Med Chem 43:4594–4605

    Article  Google Scholar 

  7. Tedesco R, Thomas JA, Katzenellenbogen BS, Katzenellenbogen JA (2001) The estrogen receptor: a structure-based approach to the design of new specific hormone-receptor combinations. Chem Biol 8:277–287

    Article  CAS  Google Scholar 

  8. Jacquot Y, Leclercq G (2009) The ligand binding domain of the human estrogen receptor alpha: mapping and functions. In: James Bartos R (ed) Estrogens: production, functions and applications. Nova, New-York, pp 231–272

    Google Scholar 

  9. Leduc AM, Trent JO, Wittliff JL et al (2003) Helix-stabilized cyclic peptides as selective inhibitors of steroid receptor–coactivator interactions. Proc Natl Acad Sci U S A 100:11273–11278

    Article  CAS  Google Scholar 

  10. Galande AK, Bramlett KS, Trent JO, Burris TP, Wittliff JL, Spatola AF (2005) Potent inhibitors of LxxLL-based protein-protein interactions. Chembiochem 6:1991–1998

    Article  CAS  Google Scholar 

  11. Singh RR, Kaluarachchi K, Chen M et al (2006) Solution structure and antiestrogenic activity of the unique C-terminal, NR-box motif-containing region of MTA1s. J Biol Chem 281:25612–25621

    Article  CAS  Google Scholar 

  12. Estebanez-Perpina E, Arnold LA, Nguyen P et al (2007) A surface of the androgen receptor that allosterically regulates coactivator binding. Proc Natl Acad Sci U S A 104:16074–16079

    Article  CAS  Google Scholar 

  13. Sivils JC, Storer CL, Galigniana MD, Cox MB (2011) Regulation of steroid hormone receptor function by the 52-kDa FK506-binding protein (FKBP52). Curr Opin Pharmacol 11:314–319

    Article  CAS  Google Scholar 

  14. De Leon JT, Iwai A, Feau C et al (2011) Targeting the regulation of androgen receptor signaling by the heat shock protein 90 cochaperone FKBP52 in prostate cancer cells. Proc Natl Acad Sci U S A 108:11878–11883

    Article  Google Scholar 

  15. Auricchio F, Migliaccio A, Castoria G, Rotondi A, Lastoria S (1984) Direct evidence of in vitro phosphorylation-dephosphorylation of the estradiol-17β receptor. Role of Ca2+-calmodulin in the activation of hormone binding sites. J Steroid Biochem 20:31–35

    Article  CAS  Google Scholar 

  16. Bouhoute A, Leclercq G (1995) Modulation of estradiol and DNA binding to estrogen receptor upon association with calmodulin. Biochem Biophys Res Commun 208:748–755

    Article  CAS  Google Scholar 

  17. Gallo D, Jacquemotte F, Cleeren A et al (2007) Calmodulin-independent, agonistic properties of a peptide containing the calmodulin binding site of estrogen receptor α. Mol Cell Endocrinol 268:37–49

    Article  CAS  Google Scholar 

  18. Gallo D, Jacquot Y, Laurent G, Leclercq G (2008) Calmodulin, a regulatory partner of the estrogen receptor alpha in breast cancer cells. Mol Cell Endocrinol 291:20–26

    Article  CAS  Google Scholar 

  19. Kelly MJ, Levin ER (2001) Rapid actions of plasma membrane estrogen receptors. Trends Endocrinol Metab 12:152–156

    Article  CAS  Google Scholar 

  20. Acconcia F, Ascenzi P, Bocedi A et al (2005) Palmitoylation-dependent estrogen receptor α membrane localization: regulation by 17β-estradiol. Mol Biol Cell 16:231–237

    Article  CAS  Google Scholar 

  21. Marino M, Ascenzi P (2006) Steroid hormone rapid signaling: the pivotal role of S-palmitoylation. IUBMB Life 58:716–719

    Article  CAS  Google Scholar 

  22. Bondar G, Kuo J, Hamid N, Micevych P (2009) Estradiol-induced estrogen receptor-α trafficking. J Neurosci 29:15323–15330

    Article  CAS  Google Scholar 

  23. Kocanova S, Mazaheri M, Caze-Subra S, Bystricky K (2010) Ligands specificity estrogen receptor alpha nuclear localization and degradation. BMC Cell Biol. doi:10.1186/1471-2121-11-98

    Article  PubMed  PubMed Central  Google Scholar 

  24. Welsh AW, Lannin DR, Young GS et al (2012) Cytoplasmic estrogen receptor in breast cancer. Clin Cancer Res 18:118–126

    Article  CAS  Google Scholar 

  25. Dull AB, George AA, Goncharova EI et al (2014) Identification of compounds by high-content screening that induce cytoplasmic to nuclear localization of a fluorescent estrogen receptor α chimera and exhibit agonist or antagonist activity in vitro. J Biomol Screen 19:242–252

    Article  CAS  Google Scholar 

  26. Peczuh MW, Hamilton AD (2000) Peptide and protein recognition by designed molecules. Chem Rev 100:2479–2494

    Article  CAS  Google Scholar 

  27. Chène P (2006) Drugs targeting protein-protein interactions. ChemMedChem 1:401–411

    Article  Google Scholar 

  28. Venyaminov SY, Yang JT (1996) Determination of protein secondary structure. In: Fasman GD (ed) Circular dichroism and the conformational analysis of biomolecules. Plenum, New York, pp 69–107

    Chapter  Google Scholar 

  29. Kelly SM, Price NC (2000) The use of circular dichroism in the investigation of protein structure and function. Curr Protein Pept Sci 1:349–384

    Article  CAS  Google Scholar 

  30. Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751:119–139

    Article  CAS  Google Scholar 

  31. Oakley MT, Bulheller BM, Hirst JD (2006) First-principles calculations of protein circular dichroism in the far-ultraviolet and beyond. Chirality 18:340–347

    Article  CAS  Google Scholar 

  32. Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1:2876–2890

    Article  CAS  Google Scholar 

  33. Bulheller BM, Rodger A, Hirst JD (2007) Circular and linear dichroism of proteins. Phys Chem Chem Phys 9:2020–2035

    Article  CAS  Google Scholar 

  34. Woody RW (2010) A significant role for high-energy transitions in the ultraviolet circular dichroism spectra of polypeptides and proteins. Chirality 22(Suppl 1):E22–E29

    Article  CAS  Google Scholar 

  35. Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A (1992) Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256:632–638

    Article  CAS  Google Scholar 

  36. Meador WE, Means AR, Quiocho FA (1992) Target enzyme recognition by calmodulin: 2.4 Å structure of a calmodulin-peptide complex. Science 257:1251–1255

    Article  CAS  Google Scholar 

  37. Barbato G, Ikura M, Kay LE, Pastor RW, Bax A (1992) Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry 31:5269–5278

    Article  CAS  Google Scholar 

  38. Meador WE, Means AR, Quiocho FA (1993) Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science 262:1718–1721

    Article  CAS  Google Scholar 

  39. Spratt DE, Taiakina V, Palmer M, Guillemette G (2007) Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes. Biochemistry 46:8288–8300

    Article  CAS  Google Scholar 

  40. Tsvetkov PO, Protasevich II, Gilli R et al (1999) Apocalmodulin binds to the myosin light chain kinase calmodulin target site. J Biol Chem 274:18161–18164

    Article  CAS  Google Scholar 

  41. Tsalkova TN, Privalov PL (1985) Thermodynamic study of domain organization in troponin C and calmodulin. J Mol Biol 181:533–544

    Article  CAS  Google Scholar 

  42. Cox JA, Comte M, Fitton JE, DeGrado WF (1985) The interaction of calmodulin with amphiphilic peptides. J Biol Chem 260:2527–2534

    CAS  PubMed  Google Scholar 

  43. Zhang M, Vogel HJ (1994) The calmodulin-binding domain of caldesmon binds to calmodulin in an α-helical conformation. Biochemistry 33:1163–1171

    Article  CAS  Google Scholar 

  44. Matsubara M, Hayashi N, Titani K, Taniguchi H (1997) Circular dichroism and 1H-NMR studies on the structures of peptides derived from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and in complex with calmodulin. J Biol Chem 272:23050–23056

    Article  CAS  Google Scholar 

  45. Yuan T, Vogel HJ (1998) Calcium-calmodulin-induced dimerization of the carboxyl-terminal domain from petunia glutamate decarboxylase. J Biol Chem 273:30328–30335

    Article  CAS  Google Scholar 

  46. Aoyagi M, Arvai AS, Tainer JA, Getzoff ED (2003) Structural basis for endothelial nitric oxide synthase binding to calmodulin. EMBO J 22:766–775

    Article  CAS  Google Scholar 

  47. Hultschig C, Hecht HJ, Frank R (2004) Systematic delineation of a calmodulin peptide interaction. J Mol Biol 343:559–568

    Article  CAS  Google Scholar 

  48. Vetter SW, Leclerc E (2003) Novel aspects of calmodulin target recognition and activation. Eur J Biochem 270:404–414

    Article  CAS  Google Scholar 

  49. Mal TK, Skrynnikov NR, Yap KL, Kay LE, Ikura M (2002) Detecting protein kinase recognition modes of calmodulin by residual dipolar couplings in solution NMR. Biochemistry 41:12899–12906

    Article  CAS  Google Scholar 

  50. Contessa GM, Orsale M, Melino S et al (2005) Structure of calmodulin complexed with an olfactory CNG channel fragment and role of the central linker: residual dipolar couplings to evaluate calmodulin binding modes outside the kinase family. J Biomol NMR 31:185–199

    Article  CAS  Google Scholar 

  51. Gallo D, Jacquot Y, Cleeren A et al (2007) Molecular basis of agonistic activity of ERα17p, a synthetic peptide corresponding to a sequence located at the N-terminal part of the estrogen receptor α ligand-binding domain. Lett Drug Des Discov 4:346–355

    Article  CAS  Google Scholar 

  52. Carlier L, Byrne C, Miclet E et al (2012) Biophysical studies of the interaction between calmodulin and the R287-T311 region of human estrogen receptor α reveals an atypical binding process. Biochem Biophys Res Commun 419:356–361

    Article  CAS  Google Scholar 

  53. Zhang M, Huque E, Vogel HJ (1994) Characterization of trimethyllysine 115 in calmodulin by 14N and 13C NMR spectroscopy. J Biol Chem 269:5099–5105

    CAS  PubMed  Google Scholar 

  54. Bourgoin-Voillard S, Fournier F, Afonso C, Jacquot Y, Leclercq G, Tabet JC (2011) Calmodulin association with the synthetic ERα17p peptide investigated by mass spectrometry. Int J Mass Spectrom 305:87–94

    Article  CAS  Google Scholar 

  55. Byrne C, Khemtémourian L, Pelekanou V et al (2012) ERα17p, a peptide reproducing the hinge region of the estrogen receptor α associates to biological membranes. Steroids 77:979–987

    Article  CAS  Google Scholar 

  56. Ruggeri FS, Byrne C, Khemtemourian L, Ducouret G, Dietler G, Jacquot Y (2015) Concentration-dependent and surface-assisted self-assembly properties of a bioactive estrogen receptor α-derived peptide. J Pept Sci 21:95–104

    Article  CAS  Google Scholar 

  57. Wallimann P, Kennedy RJ, Kemp DS (1999) Large circular dichroism ellipticities for N-templated helical polypeptides are inconsistent with currently accepted helicity algorithms. Angew Chem Int 38:1290–1292

    Article  CAS  Google Scholar 

  58. Brzozowski AM, Pike ACW, Dauter Z et al (1997) Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758

    Article  CAS  Google Scholar 

  59. Tanenbaum DM, Wang Y, Williams SP, Sigler PB (1998) Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains. Proc Natl Acad Sci U S A 95:5998–6003

    Article  CAS  Google Scholar 

  60. Jordan VC (2003) Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 2. Clinical considerations and new agents. J Med Chem 46:1081–1111

    Article  CAS  Google Scholar 

  61. Celik L, Davey J, Lund D, Schiøtt B (2008) Exploring interactions of endocrine-disrupting compounds with different conformations of the human estrogen receptor α ligand binding domain: a molecular docking study. Chem Res Toxicol 21:2195–2206

    Article  CAS  Google Scholar 

  62. Rodriguez AL, Tamrazi A, Collins ML, Katzenellenbogen JA (2004) Design, synthesis, and in vitro biological evaluation of small molecule inhibitors of estrogen receptor α coactivator binding. J Med Chem 47:600–611

    Article  CAS  Google Scholar 

  63. Gallo D, Leclercq G, Jacquot Y (2009) The N-terminal part of the ligand-binding domain of the human estrogen receptor α: a new target for estrogen disruptors. In: Colombo GP, Ricci S (eds) Medicinal chemistry research progress. Nova, New York, pp 207–224

    Google Scholar 

  64. Buzón V, Carbó LR, Estruch SB, Fletterick RJ, Estébanez-Perpiñá E (2012) A conserved surface on the ligand binding domain of nuclear receptors for allosteric control. Mol Cell Endocrinol 348:394–402

    Article  Google Scholar 

  65. Chattopadhyaya R, Meador WE, Means AR, Quiocho FA (1992) Calmodulin structure refined at 1.7 Å resolution. J Mol Biol 228:1177–1192

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to the French Ministry of Research (ED387, iViv), the Centre National pour la Recherche Scientifique (CNRS) and the Université Pierre et Marie Curie (UPMC) for financial support.

Author information

Authors and Affiliations

  1. Laboratoire des Biomolécules, Sorbonne Universités, UPMC Univ Paris 06, CNRS-UMR 7203, Université Pierre et Marie Curie-Ecole Normale Supérieure, Tour 23-33, 4, Place Jussieu, 75252, Paris Cedex 05, France

    Emeric Miclet, Cillian Byrne & Yves Jacquot

  2. Plateforme Protéomique PROMETHEE, IAB, University Grenoble Alpes-INSERM, Institut de Biologie et de Pathologie,Grenoble, France; Equipe deSpectrométrie de masse, Institut Parisien de Chimie Moléculaire, UMR 7201, UPMC Univ Paris 06, Paris, France

    Sandrine Bourgoin-Voillard

  3. INSERM, IAB, Plateforme Protéomique PROMETHEE, 38000, Grenoble, France

    Sandrine Bourgoin-Voillard

  4. CHU de Grenoble, IAB, Institut de Biologie et de Pathologie, Plateforme Protéomique PROMETHEE, 38000, Grenoble, France

    Sandrine Bourgoin-Voillard

  5. Equipe de Spectrométrie de masse, Institut Parisien de Chimie Moléculaire, UMR 7201, UPMC University Paris 06, 4, Place Jussieu, 75252, Paris Cedex 05, France

    Sandrine Bourgoin-Voillard

Authors
  1. Emeric Miclet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sandrine Bourgoin-Voillard
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cillian Byrne
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yves Jacquot
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yves Jacquot .

Editor information

Editors and Affiliations

  1. University of South Dakota Sanford School of Medicine, Vermillion, South Dakota, USA

    Kathleen M. Eyster

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Miclet, E., Bourgoin-Voillard, S., Byrne, C., Jacquot, Y. (2016). Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin. In: Eyster, K.M. (eds) Estrogen Receptors. Methods in Molecular Biology, vol 1366. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3127-9_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3127-9_19

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3126-2

  • Online ISBN: 978-1-4939-3127-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation