Skip to main content
SpringerLink
Log in

NMR analysis reveals 17β-estradiol induced conformational change in ERβ ligand binding domain expressed in E. coli

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a useful biophysical technique to study the ligand–protein interaction. In this report, we have used bacterially produced ERβ and its domains for studying the functional analysis of ligand–protein interaction. Briefly, ERβ and its transactivation domain (TAD) and ligand binding domain (LBD) were subcloned and overexpressed using a prokaryotic expression system. The recombinant proteins were purified using Ni+2-IDA affinity chromatography and analyzed by NMR. Purified ERβ and TAD show similar conformation in the absence or presence of 17β-estradiol. However, LBD shows altered conformation in the presence of 17β-estradiol. These findings suggest that ERβ produced in bacteria exhibits a conformation such that its LBD remains masked and consequently it binds less to 17β-estradiol. Such study may help to develop the therapeutic approaches for controlling the estradiol-mediated gene expression in hormone dependent diseases.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Fischer JJ, Jardetzky O (1965) Nuclear magnetic relaxation study of intermolecular complexes, the mechanism of penicillin binding to serum albumin. J Am Chem Soc 87:3237–3244

    Article  PubMed  CAS  Google Scholar 

  2. Balaram P, Bothner-By AA, Breslow E (1973) Nuclear magnetic resonance studies of the interaction of peptides and hormones with bovine neurophysin. Biochemistry 12:4695–4704

    Article  PubMed  CAS  Google Scholar 

  3. Petros AM, Ramesh V, Llinas M (1989) 1H NMR studies of aliphatic ligand binding to human plasminogen kringle 4. Biochemistry 28:1368–1376

    Article  PubMed  CAS  Google Scholar 

  4. Thewes T, Constantine K, Byeon IJ, Llinas M (1990) Ligand interactions with the kringle 5 domain of plasminogen. A study by 1H NMR spectroscopy. J Biol Chem 265:3906–3915

    PubMed  CAS  Google Scholar 

  5. Kuiper GG, Carlsson B, Grandien K, Enmark E, Hoggblad J, Nilsson S, Gustafsson JA (1997) Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 138:863–870

    Article  PubMed  CAS  Google Scholar 

  6. Gustafsson JA (1999) Estrogen receptor β-a new dimension in estrogen mechanism of action. J Endocrinol 163:379–383

    Article  PubMed  CAS  Google Scholar 

  7. Tora L, White J, Brou C, Tasset D, Webster N, Chambon P (1989) The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–487

    Article  PubMed  CAS  Google Scholar 

  8. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P (1987) Functional domains of the human estrogen receptor. Cell 51:941–951

    Article  PubMed  CAS  Google Scholar 

  9. Whitefield GK, Jurutka PW, Haussler CA, Haussler MR (1999) Steroid hormone receptors: evolution, ligands, and molecular basis of biological function. J Cell Biochem 32(33):110–122

    Article  Google Scholar 

  10. Chen JD, Evans RM (1995) A transcriptional corepressor that interacts with nuclear hormone receptors. Nature 377:454–457

    Article  PubMed  CAS  Google Scholar 

  11. Francois B (1999) Recombinant protein expression in Escherichia coli. Curr Opin Biotechnol 10:411–421

    Article  Google Scholar 

  12. Linda AL, Jessica LB, Amanda ML, Colleen HP (2000) Conformational changes in the human estrogen receptor observed by 19F NMR. Biochem Biophys Res Commun 270:988–991

    Article  Google Scholar 

  13. Jahnke W, Widmer H (2004) Protein NMR in biomedical research. Cell Mol Life Sci 61:580–599

    Article  PubMed  CAS  Google Scholar 

  14. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  15. Craik DJ, Higgins KA (1990) NMR studies of ligand macromolecule interactions. Ann Report NMR Spectrosc 22:61–138

    Article  CAS  Google Scholar 

  16. Otting G (1993) Experimental NMR techniques for studies of protein–ligand interactions. Curr Opin Struct Biol 3:760–768

    Article  CAS  Google Scholar 

  17. Hajduk PJ, Olejniczak ET, Fesik SW (1997) One-dimensional relaxation- and diffusion-edited NMR methods for screening compounds that bind to macromolecules. J Am Chem Soc 119:12257–12261

    Article  CAS  Google Scholar 

  18. Ni F (1994) Recent developments in transferred NOE methods. Prog NMR Spectrosc 26:517–606

    Article  CAS  Google Scholar 

  19. Kyle MH, Mark JN, Kathryn EC, Anobel T, Belford RL, Katzenellenbogen JA (2004) Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling. Biochemistry 43:1891–1907

    Article  Google Scholar 

  20. Takeuchi K, Wagner G (2006) NMR studies of protein interactions. Curr Opin Struc Biol 16:109–117

    Article  CAS  Google Scholar 

  21. Amadasi A, Mozzarelli A, Meda C, Maggi A, Cozzini P (2009) Identification of xenoestrogens in food additives by an integrated in silico and in vitro approach. Chem Res Toxicol 22:52–63

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We sincerely thank the financial assistance received from the Department of Biotechnology (DBT), Department of Science and Technology (DST) and Indian Council of Medical Research (ICMR), Government of India to MKT. The research fellowship from ICMR to VP is highly acknowledged.

Author information

Authors and Affiliations

  1. Department of Zoology, Biochemistry and Molecular Biology Laboratory, Centre of Advanced Study, Banaras Hindu University, Varanasi, 221005, India

    Vijay Paramanik & M. K. Thakur

Authors
  1. Vijay Paramanik
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. K. Thakur
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. K. Thakur.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paramanik, V., Thakur, M.K. NMR analysis reveals 17β-estradiol induced conformational change in ERβ ligand binding domain expressed in E. coli . Mol Biol Rep 38, 4657–4661 (2011). https://doi.org/10.1007/s11033-010-0600-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-010-0600-6

Keywords

Search

Navigation