Conformational Dynamics Analysis of MEK1 Using Hydrogen/Deuterium Exchange Mass Spectrometry

Author(s): Min Woo Yun, Kiae Kim, Ji Young Park, Ka Young Chung*

Journal Name: Protein & Peptide Letters

Volume 28 , Issue 5 , 2021


Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Abstract:

Background: Activation of mitogen-activated protein kinases (MAPKs) is regulated by a phosphorylation cascade comprising three kinases, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. MAP2K1 and MAPK2K2, also known as MEK1 and MEK2, activate ERK1 and ERK2. The structure of the MAPK signaling cascade has been studied, but high-resolution structural studies of MAP2Ks have often focused on kinase domains or docking sites, but not on full-length proteins.

Objective: To understand the conformational dynamics of MEK1.

Methods: Full-length MEK1 was purified from Escherichia coli (BL21), and its conformational dynamics were analyzed using hydrogen/deuterium exchange mass spectrometry (HDX-MS). The effects of ATP binding were examined by co-incubating MEK1 and adenylyl-imidodiphosphate (AMP- PNP), a non-hydrolysable ATP analog.

Results: MEK1 exhibited mixed EX1/EX2 HDX kinetics within the N-terminal tail through β1, αI, and the C-terminal helix. AMP-PNP binding was found to reduce conformational dynamics within the glycine-rich loop and regions near the DFG motif, along with the activation lip.

Conclusion: We report for the first time that MEK1 has regions that slowly change its folded and unfolded states (mixed EX1/EX2 kinetics) and also report the conformational effects of ATP-binding to MEK1.

Keywords: MAPK pathway, MEK1, conformational dynamics, HDX-MS, EX1, ATP.

[1]
Yang, S.H.; Sharrocks, A.D.; Whitmarsh, A.J. MAP kinase signalling cascades and transcriptional regulation. Gene, 2013, 513(1), 1-13.
[http://dx.doi.org/10.1016/j.gene.2012.10.033] [PMID: 23123731]
[2]
Morrison, D.K. MAP kinase pathways. Cold Spring Harb. Perspect. Biol., 2012, 4(11), a011254.
[http://dx.doi.org/10.1101/cshperspect.a011254] [PMID: 23125017]
[3]
Roskoski, R.Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol. Res., 2012, 66(2), 105-143.
[http://dx.doi.org/10.1016/j.phrs.2012.04.005] [PMID: 22569528]
[4]
Peti, W.; Page, R. Molecular basis of MAP kinase regulation. Protein Sci., 2013, 22(12), 1698-1710.
[http://dx.doi.org/10.1002/pro.2374] [PMID: 24115095]
[5]
Uehling, D.E.; Harris, P.A. Recent progress on MAP kinase pathway inhibitors. Bioorg. Med. Chem. Lett., 2015, 25(19), 4047-4056.
[http://dx.doi.org/10.1016/j.bmcl.2015.07.093] [PMID: 26298497]
[6]
Eishingdrelo, H.; Kongsamut, S. Minireview: Targeting GPCR activated ERK pathways for drug discovery. Curr. Chem. Genomics Transl. Med., 2013, 7, 9-15.
[http://dx.doi.org/10.2174/2213988501307010009] [PMID: 24396730]
[7]
Resing, K.A.; Ahn, N.G. Deuterium exchange mass spectrometry as a probe of protein kinase activation. Analysis of wild-type and constitutively active mutants of MAP kinase kinase-1. Biochemistry, 1998, 37(2), 463-475.
[http://dx.doi.org/10.1021/bi971750x] [PMID: 9425067]
[8]
Ohren, J.F.; Chen, H.; Pavlovsky, A.; Whitehead, C.; Zhang, E.; Kuffa, P.; Yan, C.; McConnell, P.; Spessard, C.; Banotai, C.; Mueller, W.T.; Delaney, A.; Omer, C.; Sebolt-Leopold, J.; Dudley, D.T.; Leung, I.K.; Flamme, C.; Warmus, J.; Kaufman, M.; Barrett, S.; Tecle, H.; Hasemann, C.A. Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition. Nat. Struct. Mol. Biol., 2004, 11(12), 1192-1197.
[http://dx.doi.org/10.1038/nsmb859] [PMID: 15543157]
[9]
Roskoski, R.Jr. MEK1/2 dual-specificity protein kinases: structure and regulation. Biochem. Biophys. Res. Commun., 2012, 417(1), 5-10.
[http://dx.doi.org/10.1016/j.bbrc.2011.11.145] [PMID: 22177953]
[10]
Matsumoto, T.; Kinoshita, T.; Kirii, Y.; Yokota, K.; Hamada, K.; Tada, T. Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state. Biochem. Biophys. Res. Commun., 2010, 400(3), 369-373.
[http://dx.doi.org/10.1016/j.bbrc.2010.08.071] [PMID: 20732303]
[11]
Sogabe, Y.; Hashimoto, T.; Matsumoto, T.; Kirii, Y.; Sawa, M.; Kinoshita, T. A crucial role of Cys218 in configuring an unprecedented auto-inhibition form of MAP2K7. Biochem. Biophys. Res. Commun., 2016, 473(2), 476-481.
[http://dx.doi.org/10.1016/j.bbrc.2016.03.036] [PMID: 26987717]
[12]
Glatz, G.; Gógl, G.; Alexa, A.; Reményi, A. Structural mechanism for the specific assembly and activation of the extracellular signal regulated kinase 5 (ERK5) module. J. Biol. Chem., 2013, 288(12), 8596-8609.
[http://dx.doi.org/10.1074/jbc.M113.452235] [PMID: 23382384]
[13]
Garai, Á.; Zeke, A.; Gógl, G.; Törő, I.; Fördős, F.; Blankenburg, H.; Bárkai, T.; Varga, J.; Alexa, A.; Emig, D.; Albrecht, M.; Reményi, A. Specificity of linear motifs that bind to a common mitogen-activated protein kinase docking groove. Sci. Signal., 2012, 5(245), ra74.
[http://dx.doi.org/10.1126/scisignal.2003004] [PMID: 23047924]
[14]
Min, X.; Akella, R.; He, H.; Humphreys, J.M.; Tsutakawa, S.E.; Lee, S.J.; Tainer, J.A.; Cobb, M.H.; Goldsmith, E.J. The structure of the MAP2K MEK6 reveals an autoinhibitory dimer. Structure, 2009, 17(1), 96-104.
[http://dx.doi.org/10.1016/j.str.2008.11.007] [PMID: 19141286]
[15]
Marcsisin, S.R.; Engen, J.R. Hydrogen exchange mass spectrometry: what is it and what can it tell us? Anal. Bioanal. Chem., 2010, 397(3), 967-972.
[http://dx.doi.org/10.1007/s00216-010-3556-4] [PMID: 20195578]
[16]
Lorenzen, K.; Pawson, T. HDX-MS takes centre stage at unravelling kinase dynamics. Biochem. Soc. Trans., 2014, 42(1), 145-150.
[http://dx.doi.org/10.1042/BST20130250] [PMID: 24450642]
[17]
Park, J.Y.; Yun, Y.; Chung, K.Y. Conformations of JNK3α splice variants analyzed by hydrogen/deuterium exchange mass spectrometry. J. Struct. Biol., 2017, 197(3), 271-278.
[http://dx.doi.org/10.1016/j.jsb.2016.12.005] [PMID: 27998708]
[18]
Ferraro, D.M.; Lazo, N.; Robertson, A.D. EX1 hydrogen exchange and protein folding. Biochemistry, 2004, 43(3), 587-594.
[http://dx.doi.org/10.1021/bi035943y] [PMID: 14730962]
[19]
Guttman, M.; Weis, D.D.; Engen, J.R.; Lee, K.K. Analysis of overlapped and noisy hydrogen/deuterium exchange mass spectra. J. Am. Soc. Mass Spectrom., 2013, 24(12), 1906-1912.
[http://dx.doi.org/10.1007/s13361-013-0727-5] [PMID: 24018862]
[20]
Englander, S.W.; Kallenbach, N.R. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q. Rev. Biophys., 1983, 16(4), 521-655.
[http://dx.doi.org/10.1017/S0033583500005217] [PMID: 6204354]
[21]
Weis, D.D.; Wales, T.E.; Engen, J.R.; Hotchko, M.; Ten Eyck, L.F. Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis. J. Am. Soc. Mass Spectrom., 2006, 17(11), 1498-1509.
[http://dx.doi.org/10.1016/j.jasms.2006.05.014] [PMID: 16875839]
[22]
Bereszczak, J.Z.; Watts, N.R.; Wingfield, P.T.; Steven, A.C.; Heck, A.J. Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry. Protein Sci., 2014, 23(7), 884-896.
[http://dx.doi.org/10.1002/pro.2470] [PMID: 24715628]
[23]
Fang, J.; Engen, J.R.; Beuning, P.J. Escherichia coli processivity clamp β from DNA polymerase III is dynamic in solution. Biochemistry, 2011, 50(26), 5958-5968.
[http://dx.doi.org/10.1021/bi200580b] [PMID: 21657794]
[24]
Fu, C.Y.; Prevelige, P.E.Jr. Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry. Protein Sci., 2006, 15(4), 731-743.
[http://dx.doi.org/10.1110/ps.051921606] [PMID: 16522798]
[25]
Sperry, J.B.; Ryan, Z.C.; Kumar, R.; Gross, M.L. Hydrogen/deuterium exchange reflects binding of human centrin 2 to Ca(2+) and Xeroderma pigmentosum group C peptide: an example of EX1 kinetics. Int. J. Mass Spectrom., 2012, 330-332, 302-309.
[http://dx.doi.org/10.1016/j.ijms.2012.10.013] [PMID: 23439742]
[26]
Wales, T.E.; Engen, J.R. Partial unfolding of diverse SH3 domains on a wide timescale. J. Mol. Biol., 2006, 357(5), 1592-1604.
[http://dx.doi.org/10.1016/j.jmb.2006.01.075] [PMID: 16487539]
[27]
Morgan, C.R.; Hebling, C.M.; Rand, K.D.; Stafford, D.W.; Jorgenson, J.W.; Engen, J.R. Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs. Mol. Cell Proteomics, 2011, 10(9), 010876.
[http://dx.doi.org/10.1074/mcp.M111.010876]
[28]
Duc, N.M.; Du, Y.; Zhang, C.; Lee, S.Y.; Thorsen, T.S.; Kobilka, B.K.; Chung, K.Y.; Chung, K.Y. Effective application of bicelles for conformational analysis of G protein-coupled receptors by hydrogen/deuterium exchange mass spectrometry. J. Am. Soc. Mass Spectrom., 2015, 26(5), 808-817.
[http://dx.doi.org/10.1007/s13361-015-1083-4] [PMID: 25740347]
[29]
Fang, J.; Rand, K.D.; Beuning, P.J.; Engen, J.R. False EX1 signatures caused by sample carryover during HX MS analyses. Int. J. Mass Spectrom., 2011, 302(1-3), 19-25.
[http://dx.doi.org/10.1016/j.ijms.2010.06.039] [PMID: 21643454]
[30]
Owen, G.R.; Stoychev, S.; Achilonu, I.; Dirr, H.W. Phosphorylation- and nucleotide-binding-induced changes to the stability and hydrogen exchange patterns of JNK1β1 provide insight into its mechanisms of activation. J. Mol. Biol., 2014, 426(21), 3569-3589.
[http://dx.doi.org/10.1016/j.jmb.2014.08.019] [PMID: 25178256]
[31]
Ring, A.Y.; Sours, K.M.; Lee, T.; Ahn, N.G. Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2. Int. J. Mass Spectrom., 2011, 302(1-3), 101-109.
[http://dx.doi.org/10.1016/j.ijms.2010.08.020] [PMID: 21785572]
[32]
Sours, K.M.; Kwok, S.C.; Rachidi, T.; Lee, T.; Ring, A.; Hoofnagle, A.N.; Resing, K.A.; Ahn, N.G. Hydrogen-exchange mass spectrometry reveals activation-induced changes in the conformational mobility of p38alpha MAP kinase. J. Mol. Biol., 2008, 379(5), 1075-1093.
[http://dx.doi.org/10.1016/j.jmb.2008.04.044] [PMID: 18501927]
[33]
Lee, T.; Hoofnagle, A.N.; Resing, K.A.; Ahn, N.G. Hydrogen exchange solvent protection by an ATP analogue reveals conformational changes in ERK2 upon activation. J. Mol. Biol., 2005, 353(3), 600-612.
[http://dx.doi.org/10.1016/j.jmb.2005.08.029] [PMID: 16185715]
[34]
Lee, T.; Hoofnagle, A.N.; Kabuyama, Y.; Stroud, J.; Min, X.; Goldsmith, E.J.; Chen, L.; Resing, K.A.; Ahn, N.G. Docking motif interactions in MAP kinases revealed by hydrogen exchange mass spectrometry. Mol. Cell, 2004, 14(1), 43-55.
[http://dx.doi.org/10.1016/S1097-2765(04)00161-3] [PMID: 15068802]
[35]
Hoofnagle, A.N.; Resing, K.A.; Goldsmith, E.J.; Ahn, N.G. Changes in protein conformational mobility upon activation of extracellular regulated protein kinase-2 as detected by hydrogen exchange. Proc. Natl. Acad. Sci. USA, 2001, 98(3), 956-961.
[http://dx.doi.org/10.1073/pnas.98.3.956] [PMID: 11158577]
[36]
Mansour, S.J.; Candia, J.M.; Matsuura, J.E.; Manning, M.C.; Ahn, N.G. Interdependent domains controlling the enzymatic activity of mitogen-activated protein kinase kinase 1. Biochemistry, 1996, 35(48), 15529-15536.
[http://dx.doi.org/10.1021/bi961854s] [PMID: 8952507]
[37]
Park, E.; Rawson, S.; Li, K.; Kim, B.W.; Ficarro, S.B.; Pino, G.G.; Sharif, H.; Marto, J.A.; Jeon, H.; Eck, M.J. Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes. Nature, 2019, 575(7783), 545-550.
[http://dx.doi.org/10.1038/s41586-019-1660-y] [PMID: 31581174]
[38]
Fischmann, T.O.; Smith, C.K.; Mayhood, T.W.; Myers, J.E.; Reichert, P.; Mannarino, A.; Carr, D.; Zhu, H.; Wong, J.; Yang, R.S.; Le, H.V.; Madison, V.S. Crystal structures of MEK1 binary and ternary complexes with nucleotides and inhibitors. Biochemistry, 2009, 48(12), 2661-2674.
[http://dx.doi.org/10.1021/bi801898e] [PMID: 19161339]
[39]
Brennan, D.F.; Dar, A.C.; Hertz, N.T.; Chao, W.C.; Burlingame, A.L.; Shokat, K.M.; Barford, D. A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK. Nature, 2011, 472(7343), 366-369.
[http://dx.doi.org/10.1038/nature09860] [PMID: 21441910]
[40]
Ki, A.Y.; Park, J.Y.; Chung, K.Y. Conformational changes of JNK3 splice variants upon ATP binding. Biodesign, 2019, 7(1), 1-5.


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 28
ISSUE: 5
Year: 2021
Published on: 03 November, 2020
Page: [481 - 488]
Pages: 8
DOI: 10.2174/0929866527666201103152534
Price: $65

Article Metrics

PDF: 44
HTML: 4