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Hydrogen-Deuterium Exchange and Hydroxyl Radical Footprinting for Mapping Hydrophobic Interactions of Human Bromodomain with a Small Molecule Inhibitor

  • Ke Sherry Li
  • Elizabeth T. Schaper Bergman
  • Brett R. Beno
  • Richard Y.-C. Huang
  • Ekaterina Deyanova
  • Guodong Chen
  • Michael L. GrossEmail author
Research Article

Abstract

Mass spectrometry (MS)–based protein footprinting, a valuable structural tool in mapping protein-ligand interaction, has been extensively applied to protein-protein complexes, showing success in mapping large interfaces. Here, we utilized an integrated footprinting strategy incorporating both hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (i.e., fast photochemical oxidation of proteins (FPOP)) for molecular-level characterization of the interaction of human bromodomain-containing protein 4 (BRD4) with a hydrophobic benzodiazepine inhibitor. HDX does not provide strong evidence for the location of the binding interface, possibly because the shielding of solvent by the small molecule is not large. Instead, HDX suggests that BRD4 appears to be stabilized by showing a modest decrease in dynamics caused by binding. In contrast, FPOP points to a critical binding region in the hydrophobic cavity, also identified by crystallography, and, therefore, exhibits higher sensitivity than HDX in mapping the interaction of BRD4 with compound 1. In the absence or under low concentrations of the radical scavenger, FPOP modifications on Met residues show significant differences that reflect the minor change in protein conformation. This problem can be avoided by using a sufficient amount of proper scavenger, as suggested by the FPOP kinetics directed by a dosimeter of the hydroxyl radical.

Keywords

Protein Small-molecule binding Mass spectrometry Fast photochemical oxidation of proteins (FPOP) Hydrogen-deuterium exchange Benzyl (1-methyl-6-phenyl-4Hbenzo[f][1,2,4]triazolo[4,3-a][1,4]diazepin-4-yl)carbamate Human bromodomain-containing protein 4 (BRD4) 

Notes

Acknowledgements

This research was supported by the NIH (Grant P41GM103422) and by a Research Collaboration with Bristol-Myers Squibb. The authors thank Dr. Olafur Gudmundsson and Dr. Lois Lehman-McKeeman from Bristol-Myers Squibb for their support of this project. The authors would also like to thank Frank Marsilio, Chunhong Yan, and Dr. Ashok Purandare from Bristol-Myers Squibb for technical assistance.

Supplementary material

13361_2019_2316_MOESM1_ESM.docx (1.3 mb)
ESM 1 (DOCX 1361 kb)

References

  1. 1.
    Cohen, Y.: Small Molecules: the Silent Majority of Pharmaceutical Pipelines. (2015)Google Scholar
  2. 2.
    Masson, G.R., Jenkins, M.L., Burke, J.E.: An overview of hydrogen deuterium exchange mass spectrometry (HDX-MS) in drug discovery. Expert Opin. Drug Discovery. 12, 981–994 (2017)CrossRefGoogle Scholar
  3. 3.
    Pirrone, G.F., Iacob, R.E., Engen, J.R.: Applications of hydrogen/deuterium exchange MS from 2012 to 2014. Anal. Chem. 87, 99–118 (2015)CrossRefGoogle Scholar
  4. 4.
    Engen, J.R.: Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS. Anal. Chem. 81, 7870–7875 (2009)CrossRefGoogle Scholar
  5. 5.
    Huang, R.Y.C., O’Neil, S.R., Lipovšek, D., Chen, G.: Conformational assessment of adnectin and adnectin-drug conjugate by hydrogen/deuterium exchange mass spectrometry. J. Am. Soc. Mass Spectrom. 29, 1524–1531 (2018)CrossRefGoogle Scholar
  6. 6.
    Canetti, D., Rendell, N.B., Di Vagno, L., Gilbertson, J.A., Rowczenio, D., Rezk, T., Gillmore, J.D., Hawkins, P.N., Verona, G., Mangione, P.P., Giorgetti, S., Mauri, P., Motta, S., De Palma, A., Bellotti, V., Taylor, G.W.: Misidentification of transthyretin and immunoglobulin variants by proteomics due to methyl lysine formation in formalin-fixed paraffin-embedded amyloid tissue. Amyloid. 24, 233–241 (2017)CrossRefGoogle Scholar
  7. 7.
    Gu, X., Yan, Y., Novick, S.J., Kovach, A., Goswami, D., Ke, J., Tan, M.H.E., Wang, L., Li, X., De Waal, P.W., Webb, M.R., Griffin, P.R., Xu, H.E., Melcher, K.: Deconvoluting AMP-activated protein kinase (AMPK) adenine nucleotide binding and sensing. J. Biol. Chem. 292, 12653–12666 (2017)CrossRefGoogle Scholar
  8. 8.
    Zorba, A., Nguyen, C., Xu, Y., Starr, J., Borzilleri, K., Smith, J., Zhu, H., Farley, K.A., Ding, W., Schiemer, J., Feng, X., Chang, J.S., Uccello, D.P., Young, J.A., Garcia-Irrizary, C.N., Czabaniuk, L., Schuff, B., Oliver, R., Montgomery, J., Hayward, M.M., Coe, J., Chen, J., Niosi, M., Luthra, S., Shah, J.C., El-Kattan, A., Qiu, X., West, G.M., Noe, M.C., Shanmugasundaram, V., Gilbert, A.M., Brown, M.F., Calabrese, M.F.: Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc. Natl. Acad. Sci. U. S. A. 115, E7285–E7292 (2018)CrossRefGoogle Scholar
  9. 9.
    Marciano, D.P., Dharmarajan, V., Griffin, P.R.: HDX-MS guided drug discovery: small molecules and biopharmaceuticals. Curr. Opin. Struct. Biol. 28, 105–111 (2014)CrossRefGoogle Scholar
  10. 10.
    Dai, S.Y., Chalmers, M.J., Bruning, J., Bramlett, K.S., Osborne, H.E., Montrose-Rafizadeh, C., Barr, R.J., Wang, Y., Wang, M., Burris, T.P., Dodge, J.A., Griffin, P.R.: Prediction of the tissue-specificity of selective estrogen receptor modulators by using a single biochemical method. Proc. Natl. Acad. Sci. 105, 7171-7176 (2008)Google Scholar
  11. 11.
    Hernychova, L., Man, P., Verma, C., Nicholson, J., Sharma, C.A., Ruckova, E., Teo, J.Y., Ball, K., Vojtesek, B., Hupp, T.R.: Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry. Proteomics. 13, 2512–2525 (2013)CrossRefGoogle Scholar
  12. 12.
    Wang, H., Rempel, D.L., Giblin, D., Frieden, C., Gross, M.L.: Peptide-level interactions between proteins and small-molecule drug candidates by two hydrogen−deuterium exchange MS-based methods: the example of apolipoprotein E3. Anal. Chem. 89, 10687–10695 (2017)CrossRefGoogle Scholar
  13. 13.
    Xu, G., Chance, M.R.: Hydroxyl radical-mediated modification of proteins as probes for structural proteomics. Chem. Rev. 107, 3514–3543 (2007)CrossRefGoogle Scholar
  14. 14.
    Hambly, D.M., Gross, M.L.: Laser flash photolysis of hydrogen peroxide to oxidize protein solvent-accessible residues on the microsecond timescale. J. Am. Soc. Mass Spectrom. 16, 2057–2063 (2005)CrossRefGoogle Scholar
  15. 15.
    Li, K.S., Shi, L., Gross, M.L.: Mass spectrometry-based fast photochemical oxidation of proteins (FPOP) for higher order structure characterization. Acc. Chem. Res. 51, 736–744 (2018)CrossRefGoogle Scholar
  16. 16.
    Gau, B.C., Sharp, J.S., Rempel, D.L., Gross, M.L.: Fast photochemical oxidation of protein footprints faster than protein unfolding. Anal. Chem. 81, 6563–6571 (2009)CrossRefGoogle Scholar
  17. 17.
    Li, K.S., Rempel, D.L., Gross, M.L.: Conformational-sensitive fast photochemical oxidation of proteins and mass spectrometry characterize amyloid beta 1–42 aggregation. J. Am. Chem. Soc. 138, 12090–12098 (2016)CrossRefGoogle Scholar
  18. 18.
    Chen, J., Rempel, D.L., Gau, B.C., Gross, M.L.: Fast photochemical oxidation of proteins and mass spectrometry follow submillisecond protein folding at the amino-acid level. J. Am. Chem. Soc. 134, 18724–18731 (2012)CrossRefGoogle Scholar
  19. 19.
    Zhang, H., Gau, B.C., Jones, L.M., Vidavsky, I., Gross, M.L.: Fast photochemical oxidation of proteins (FPOP) for comparing structures of protein/ligand complexes: the calmodulin-peptide model system. Anal. Chem. 83, 311–318 (2011)CrossRefGoogle Scholar
  20. 20.
    Zhang, Y., Wecksler, A.T., Molina, P., Deperalta, G., Gross, M.L.: Mapping the binding interface of VEGF and a monoclonal antibody Fab-1 fragment with fast photochemical oxidation of proteins (FPOP) and mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 850-858 (2017)Google Scholar
  21. 21.
    Li, K.S., Chen, G., Mo, J., Huang, R.Y.C., Deyanova, E.G., Beno, B.R., O’Neil, S.R., Tymiak, A.A., Gross, M.L.: Orthogonal mass spectrometry-based footprinting for epitope mapping and structural characterization: the IL-6 receptor upon binding of protein therapeutics. Anal. Chem. 89, 7742–7749 (2017)CrossRefGoogle Scholar
  22. 22.
    Yan, Y., Chen, G., Wei, H., Huang, R.Y.-C., Mo, J., Rempel, D.L., Tymiak, A.A., Gross, M.L.: Fast photochemical oxidation of proteins (FPOP) maps the epitope of EGFR binding to adnectin. J. Am. Soc. Mass Spectrom. 25, 2084–2092 (2014)CrossRefGoogle Scholar
  23. 23.
    Jones, L.M., Sperry, J., Carroll, J., Gross, M.L.: Fast photochemical oxidation of proteins for epitope mapping. Anal. Chem. 83, 7657–7661 (2011)CrossRefGoogle Scholar
  24. 24.
    Li, J., Chen, G.: The use of fast photochemical oxidation of proteins coupled with mass spectrometry in protein therapeutics discovery and development. Drug Discov. Today, 24, 829-834 (2019)Google Scholar
  25. 25.
    Filippakopoulos, P., Picaud, S., Mangos, M., Keates, T., Lambert, J.P., Barsyte-Lovejoy, D., Felletar, I., Volkmer, R., Muller, S., Pawson, T., Gingras, A.C., Arrowsmith, C.H., Knapp, S.: Histone recognition and large-scale structural analysis of the human bromodomain family. Cell. 149, 214–231 (2012)CrossRefGoogle Scholar
  26. 26.
    Chung, C.W., Coste, H., White, J.H., Mirguet, O., Wilde, J., Gosmini, R.L., Delves, C., Magny, S.M., Woodward, R., Hughes, S.A., Boursier, E.V., Flynn, H., Bouillot, A.M., Bamborough, P., Brusq, J.M., Gellibert, F.J., Jones, E.J., Riou, A.M., Homes, P., Martin, S.L., Uings, I.J., Toum, J., Clement, C.A., Boullay, A.B., Grimley, R.L., Blandel, F.M., Prinjha, R.K., Lee, K., Kirilovsky, J., Nicodeme, E.: Discovery and characterization of small molecule inhibitors of the BET family bromodomains. J. Med. Chem. 54, 3827–3838 (2011)CrossRefGoogle Scholar
  27. 27.
    Malito, E., Faleri, A., Lo Surdo, P., Veggi, D., Maruggi, G., Grassi, E., Cartocci, E., Bertoldi, I., Genovese, A., Santini, L., Romagnoli, G., Borgogni, E., Brier, S., Lo Passo, C., Domina, M., Castellino, F., Felici, F., van der Veen, S., Johnson, S., Lea, S.M., Tang, C.M., Pizza, M., Savino, S., Norais, N., Rappuoli, R., Bottomley, M.J., Masignani, V.: Defining a protective epitope on factor H binding protein, a key meningococcal virulence factor and vaccine antigen. Proc Natl Acad Sci U S A. 110, 3304–3309 (2013)CrossRefGoogle Scholar
  28. 28.
    Zhang, J., Adrián, F.J., Jahnke, W., Cowan-Jacob, S.W., Li, A.G., Iacob, R.E., Sim, T., Powers, J., Dierks, C., Sun, F., Guo, G.-R., Ding, Q., Okram, B., Choi, Y., Wojciechowski, A., Deng, X., Liu, G., Fendrich, G., Strauss, A., Vajpai, N., Grzesiek, S., Tuntland, T., Liu, Y., Bursulaya, B., Azam, M., Manley, P.W., Engen, J.R., Daley, G.Q., Warmuth, M., Gray, N.S.: Targeting Bcr–Abl by combining allosteric with ATP-binding-site inhibitors. Nature. 463, 501 (2010)CrossRefGoogle Scholar
  29. 29.
    Brudler, R., Gessner, C.R., Li, S., Tyndall, S., Getzoff, E.D., Woods Jr., V.L.: PAS domain allostery and light-induced conformational changes in photoactive yellow protein upon I2 intermediate formation, probed with enhanced hydrogen/deuterium exchange mass spectrometry. J. Mol. Biol. 363, 148–160 (2006)CrossRefGoogle Scholar
  30. 30.
    Niu, B., Mackness, B.C., Rempel, D.L., Zhang, H., Cui, W., Matthews, C.R., Zitzewitz, J.A., Gross, M.L.: Incorporation of a reporter peptide in FPOP compensates for adventitious scavengers and permits time-dependent measurements. J. Am. Soc. Mass Spectrom. 28, 389–392 (2017)CrossRefGoogle Scholar
  31. 31.
    Xie, B., Sood, A., Woods, R.J., Sharp, J.S.: Quantitative protein topography measurements by high resolution hydroxyl radical protein footprinting enable accurate molecular model selection. Sci. Rep. 7, 4552 (2017)CrossRefGoogle Scholar
  32. 32.
    Shi, L., Liu, T., Gross, M.L., Huang, Y.: Recognition of human IgG1 by Fcγ receptors: structural insights from hydrogen–deuterium exchange and fast photochemical oxidation of proteins coupled with mass spectrometry. Biochemistry. 58, 1074–1080 (2019)CrossRefGoogle Scholar
  33. 33.
    Flynn, E.M., Huang, O.W., Poy, F., Oppikofer, M., Bellon, S.F., Tang, Y., Cochran, A.G.: A subset of human bromodomains recognizes butyryllysine and crotonyllysine histone peptide modifications. Structure. 23, 1801–1814 (2015)CrossRefGoogle Scholar
  34. 34.
    Ember, S.W.J., Zhu, J.-Y., Olesen, S.H., Martin, M.P., Becker, A., Berndt, N., Georg, G.I., Schönbrunn, E.: Acetyl-lysine binding site of bromodomain-containing protein 4 (BRD4) interacts with diverse kinase inhibitors. ACS Chem. Biol. 9, 1160–1171 (2014)CrossRefGoogle Scholar
  35. 35.
    Li, Z., Moniz, H., Wang, S., Ramiah, A., Zhang, F., Moremen, K.W., Linhardt, R.J., Sharp, J.S.: High structural resolution hydroxyl radical protein footprinting reveals an extended Robo1-heparin binding interface. J. Biol. Chem. 290, 10729–10740 (2015)CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2019

Authors and Affiliations

  1. 1.Department of ChemistryWashington UniversitySt. LouisUSA
  2. 2.Molecular Structure & Design, Research and DevelopmentBristol-Myers SquibbPrincetonUSA
  3. 3.Pharmaceutical Candidate Optimization, Research and DevelopmentBristol-Myers SquibbPrincetonUSA

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