Skip to main content
SpringerLink
Log in

Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

An interplay between monomeric and dimeric forms of human epidermal growth factor (EGF) affecting its interaction with EGF receptor (EGFR) is poorly understood. While EGF dimeric structure was resolved at pH 8.1, the possibility of EGF dimerization under physiological conditions is still unclear. This study aimed to describe the oligomeric state of EGF in a solution at physiological pH value. With centrifugal ultrafiltration followed by blue native gel electrophoresis, we showed that synthetic human EGF in a solution at a concentration of 0.1 mg/ml exists mainly in the dimeric form at pH 7.4 and temperature of 37 °C, although a small fraction of its monomers was also observed. Based on bioinformatics predictions, we introduced the D46G substitution to examine if EGF C-terminal part is directly involved in the intermolecular interface formation of the observed dimers. We found a reduced ability of the resulting EGF D46G dimers to dissociate at temperatures up to 50 °C. The D46G substitution also increased the intermolecular antiparallel β-structure content within the EGF peptide in a solution according to the CD spectra analysis that was confirmed by HATR-FTIR results. Additionally, the energy transfer between Tyr and Trp residues was detected by fluorescence spectroscopy for the EGF D46G mutant, but not for the native EGF. This allowed us to suggest the elongation and rearrangement of the intermolecular β-structure that leads to the observed stabilization of EGF D46G dimers. The results imply EGF dimerization under physiological pH value and temperature and the involvement of EGF C-terminal part in this process.

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
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Code Availability

Not applicable.

Abbreviations

BN-PAGE:

Blue native polyacrylamide gel electrophoresis

EGF:

Epidermal growth factor

EGFR:

Epidermal growth factor receptor

CD:

Circular dichroism;

MWCO:

Molecular weight cut-off

NMR:

Nuclear magnetic resonance

PB:

Phosphate buffer

HATR-FTIR:

Horizontal attenuated total reflection Fourier transform infrared spectroscopy

References

  1. Carpenter G, Cohen S (1979) Epidermal growth factor. Annu Rev Biochem 48:193–216. https://doi.org/10.1146/annurev.bi.48.070179.001205

    Article  CAS  PubMed  Google Scholar 

  2. Lu HS, Chai JJ, Li M, Huang BR, He CH, Bi RC (2001) Crystal structure of human epidermal growth factor and its dimerization. J Biol Chem 276(37):34913–34917. https://doi.org/10.1074/jbc.M102874200

    Article  CAS  PubMed  Google Scholar 

  3. Huang HW, Mohan SK, Yu C (2010) The NMR solution structure of human epidermal growth factor (hEGF) at physiological pH and its interactions with suramin. Biochem Biophys Res Commun 402(4):705–710. https://doi.org/10.1016/j.bbrc.2010.10.089

    Article  CAS  PubMed  Google Scholar 

  4. Zanetti-Domingues LC, Korovesis D, Needham SR, Tynan CJ, Sagawa S, Roberts SK et al (2018) The architecture of EGFR’s basal complexes reveals autoinhibition mechanisms in dimers and oligomers. Nat Commun 9(1):4325. https://doi.org/10.1038/s41467-018-06632-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Yu X, Sharma KD, Takahashi T, Iwamoto R, Mekada E (2002) Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling. Mol Biol Cell 13(7):2547–2557. https://doi.org/10.1091/mbc.01-08-0411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Marianayagam NJ, Sunde M, Matthews JM (2004) The power of two: protein dimerization in biology. Trends Biochem Sci 29(11):618–625. https://doi.org/10.1016/j.tibs.2004.09.006

    Article  CAS  PubMed  Google Scholar 

  7. Lu C, Mi LZ, Grey MJ, Zhu J, Graef E, Yokoyama S et al (2010) Structural evidence for loose linkage between ligand binding and kinase activation in the epidermal growth factor receptor. Mol Cell Biol 30(22):5432–5443. https://doi.org/10.1128/MCB.00742-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M, Kim JH et al (2002) Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110(6):775–787. https://doi.org/10.1016/s0092-8674(02)00963-7

    Article  CAS  PubMed  Google Scholar 

  9. Gallay J, Vincent M, Li de la Sierra IM, Alvarez J, Ubieta R, Madrazo J et al (1993) Protein flexibility and aggregation state of human epidermal growth factor. A time-resolved fluorescence study of the native protein and engineered single-tryptophan mutants. Eur J Biochem 211(1–2):213–219. https://doi.org/10.1111/j.1432-1033.1993.tb19888.x

    Article  CAS  PubMed  Google Scholar 

  10. Jacob J, Duclohier H, Cafiso DS (1999) The role of proline and glycine in determining the backbone flexibility of a channel-forming peptide. Biophys J 76(3):1367–1376. https://doi.org/10.1016/S0006-3495(99)77298-X

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wittig I, Braun HP, Schägger H (2006) Blue native PAGE. Nat Protoc 1(1):418–428. https://doi.org/10.1038/nprot.2006.62

    Article  CAS  PubMed  Google Scholar 

  12. Hirota S, Hattori Y, Nagao S, Taketa M, Komori H, Kamikubo H et al (2010) Cytochrome c polymerization by successive domain swapping at the C-terminal helix. Proc Natl Acad Sci USA 107(29):12854–12859. https://doi.org/10.1073/pnas.1001839107

    Article  PubMed  PubMed Central  Google Scholar 

  13. Kozlowski LP (2021) IPC 2.0: prediction of isoelectric point and pKa dissociation constants. Nucleic Acids Res 49(W1):W285–W292. https://doi.org/10.1093/nar/gkab295

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Micsonai A, Wien F, Bulyáki É, Kun J, Moussong E, Lee YH et al (2018) BeStSel: a web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra. Nucleic Acids Res 46(W1):W315–W322. https://doi.org/10.1093/nar/gky497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New York, p 954

    Book  Google Scholar 

  16. Broos J, Tveen-Jensen K, de Waal E, Hesp BH, Jackson JB, Canters GW et al (2007) The emitting state of tryptophan in proteins with highly blue-shifted fluorescence. Angew Chem Int Ed Engl 46(27):5137–5139. https://doi.org/10.1002/anie.200700839

    Article  CAS  PubMed  Google Scholar 

  17. Yang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43(W1):W174–W181. https://doi.org/10.1093/nar/gkv342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Xu D, Zhang Y (2013) Toward optimal fragment generations for ab initio protein structure assembly. Proteins 81(2):229–239. https://doi.org/10.1002/prot.24179

    Article  CAS  PubMed  Google Scholar 

  19. Shen Y, Maupetit J, Derreumaux P, Tufféry P (2014) Improved PEP-fold approach for peptide and miniprotein structure prediction. J Chem Theory Comput 10(10):4745–4758. https://doi.org/10.1021/ct500592m

    Article  CAS  PubMed  Google Scholar 

  20. Deng H, Jia Y, Zhang Y (2016) 3Drobot: automated generation of diverse and well-packed protein structure decoys. Bioinformatics 32(3):378–387. https://doi.org/10.1093/bioinformatics/btv601

    Article  CAS  PubMed  Google Scholar 

  21. Doig AJ, MacArthur MW, Stapley BJ, Thornton JM (1997) Structures of N-termini of helices in proteins. Protein Sci 6(1):147–155. https://doi.org/10.1002/pro.5560060117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Khrustalev VV, Khrustaleva TA, Poboinev VV, Stojarov AN, Kordyukova LV, Akunevich AA (2021) Spectra of tryptophan fluorescence are the result of co-existence of certain most abundant stabilized excited state and certain most abundant destabilized excited state. Spectrochim Acta A Mol Biomol Spectrosc 257:119784. https://doi.org/10.1016/j.saa.2021.119784

    Article  CAS  Google Scholar 

  23. Hristova SH, Zhivkov AM (2019) Isoelectric point of free and adsorbed cytochrome c determined by various methods. Colloids Surf B Biointerfaces 174:87–94. https://doi.org/10.1016/j.colsurfb.2018.10.080

    Article  CAS  PubMed  Google Scholar 

  24. Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1(6):2876–2890. https://doi.org/10.1038/nprot.2006.202

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Whitmore L, Wallace BA (2008) Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 89(5):392–400. https://doi.org/10.1002/bip.20853

    Article  CAS  PubMed  Google Scholar 

  26. Sadat A, Joye IJ (2020) Peak fitting applied to fourier transform infrared and Raman spectroscopic analysis of proteins. Appl Sci 10:5918. https://doi.org/10.3390/app10175918

    Article  CAS  Google Scholar 

  27. Barth A (2007) Infrared spectroscopy of proteins. Biochim Biophys Acta 1767:1073–1101. https://doi.org/10.1016/j.bbabio.2007.06.004

    Article  CAS  PubMed  Google Scholar 

  28. Vivian JT, Callis PR (2001) Mechanisms of tryptophan fluorescence shifts in proteins. Biophys J 80(5):2093–2109. https://doi.org/10.1016/S0006-3495(01)76183-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Teng Q (2005) Structural biology: practical NMR applications. Springer, New York, p 295

    Google Scholar 

  30. Ferguson KM, Berger MB, Mendrola JM, Cho HS, Leahy DJ, Lemmon MA (2003) EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization. Mol Cell 11(2):507–517. https://doi.org/10.1016/s1097-2765(03)00047-9

    Article  CAS  PubMed  Google Scholar 

  31. Marqueze-Pouey B, Mailfert S, Rouger V, Goaillard JM, Marguet D (2014) Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations. PLoS ONE 9(9):e106803. https://doi.org/10.1371/journal.pone.0106803

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Khrustalev VV, Khrustaleva TA, Kahanouskaya EY, Rudnichenko YA, Bandarenka HV, Arutyunyan AM et al (2018) The alpha helix 1 from the first conserved region of HIV1 gp120 is reconstructed in the short NQ21 peptide. Arch Biochem Biophys 638:66–75. https://doi.org/10.1016/j.abb.2017.12.004

    Article  CAS  PubMed  Google Scholar 

  33. Carugo O (2014) Buried chloride stereochemistry in the Protein Data Bank. BMC Struct Biol 14:19. https://doi.org/10.1186/s12900-014-0019-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by the Belarusian Republican Foundation for Fundamental Research [Grant Number B20M-025].

Author information

Authors and Affiliations

  1. Department of General Chemistry, Belarusian State Medical University, Dzerzhinskogo 83, 220045, Minsk, Belarus

    Anastasia Aleksandrovna Akunevich, Vladislav Victorovich Khrustalev, Victor Vitoldovich Poboinev & Nikolai Vladimirovich Shalygo

  2. Laboratory of Multiprofile Diagnostics, Institute of Physiology of the National Academy of Sciences of Belarus, Academicheskaya 28, 220072, Minsk, Belarus

    Tatyana Aleksandrovna Khrustaleva

  3. Department of Radiation Medicine and Ecology, Belarusian State Medical University, Dzerzhinskogo 83, 220045, Minsk, Belarus

    Aleksander Nicolaevich Stojarov

  4. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1-40, Moscow, Russia, 119991

    Alexander Migranovich Arutyunyan & Larisa Valentinovna Kordyukova

  5. Centre for Physical and Chemical Investigation Methods, Belarusian State Technological University, Sverdlova 13a, 220006, Minsk, Belarus

    Yehor Gennadyevich Sapon

Authors
  1. Anastasia Aleksandrovna Akunevich
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vladislav Victorovich Khrustalev
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tatyana Aleksandrovna Khrustaleva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Victor Vitoldovich Poboinev
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nikolai Vladimirovich Shalygo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Aleksander Nicolaevich Stojarov
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexander Migranovich Arutyunyan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Larisa Valentinovna Kordyukova
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yehor Gennadyevich Sapon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AAA: Investigation, Formal analysis, Writing—Original draft preparation, Visualization. VVK: Conceptualization, Investigation, Formal analysis, Writing—Original draft preparation. TAK: Conceptualization, Investigation, Supervision, Writing—Review and editing. VVP: Investigation, Formal analysis, Writing—Review and editing. NVS: Methodology, Investigation, Writing—Review and editing. ANS: Methodology, Investigation, Writing—Review and editing. AMA: Investigation, Writing—Review and editing. LVK: Investigation, Writing—Review and editing. YGS: Methodology, Investigation.

Corresponding author

Correspondence to Anastasia Aleksandrovna Akunevich.

Ethics declarations

Conflict of interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (XLSX 18 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Akunevich, A.A., Khrustalev, V.V., Khrustaleva, T.A. et al. Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution. Protein J 41, 245–259 (2022). https://doi.org/10.1007/s10930-022-10051-y

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-022-10051-y

Keywords

Search

Navigation