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Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions

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Abstract

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19F-labeling readily provided NMR detection of protein-drug and protein–protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19F-labeling method was 3.5-fold more sensitive than 15N-labeling, and could be combined with other chemical modification techniques such as lysine 13C-methylation. 13C-dimethylated-19F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

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References

  • Ando H, Adachi M, Umeda K, Matsuura A, Nonaka M, Uchio R, Tanaka H, Motoki M (2014) Purification and characteristics of a novel transglutaminase derived from microorganisms. Agr Biol Chem 53:2613–2617

    Google Scholar 

  • Aramini JM, Hamilton K, Ma L-C, Swapna GVT, Leonard PG, Ladbury JE, Krug RM, Montelione GT (2014) 19F NMR reveals multiple conformations at the dimer interface of the non-structural protein 1 effector domain from influenza A virus. Structure 22:515–525

    Article  Google Scholar 

  • Arntson KE, Pomerantz WCK (2016) Protein-observed fluorine NMR a bioorthogonal approach for small molecule discovery. J Med Chem 59:5158–5171

    Article  Google Scholar 

  • Barden JA, Phillips L, Cornell BA, Dos Remedios CG (2002) Fluorine-19 NMR studies of the interaction of selectively labeled actin and myosin. Biochemistry 28:5895–5901

    Article  Google Scholar 

  • Bokoch MP, Zou Y, Rasmussen SGF, Liu CW, Nygaard R, Rosenbaum DM, Fung JJ, Choi H-J, Thian FS, Kobilka TS, Puglisi JD, Weis WI, Pardo L, Prosser RS, Mueller L, Kobilka BK (2010) Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463:108–112

    Article  ADS  Google Scholar 

  • Cavanagh J, Fairbrother WJ, Palmer AG, Rance M, Skelton NJ (2007) Protein NMR spectroscopy. Academic Press, Burlington

    Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  Google Scholar 

  • Folk JE (1980) Transglutaminases. Annu Rev Biochem 49:517–531

    Article  Google Scholar 

  • Fontana A, Spolaore B, Mero A, Veronese FM (2008) Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase. Adv Drug Deliver Rev 60:13–28

    Article  Google Scholar 

  • Gee CT, Koleski EJ, Pomerantz WCK (2015) Fragment screening and druggability assessment for the CBP/p300 KIX domain through protein-observed 19F NMR spectroscopy. Angew Chem Int Ed 54:3735–3739

    Article  Google Scholar 

  • Gerig JT (1994) Fluorine NMR of proteins. Prog Nucl Magn Reson Spectrosc 26:293–370

    Article  Google Scholar 

  • Goddard TD, Kneller DG (2008) SPARKY 3.

  • Hattori Y, Furuita K, Ohki I, Ikegami T, Fukada H, Shirakawa M, Fujiwara T, Kojima C (2013) Utilization of lysine 13C-methylation NMR for protein–protein interaction studies. J Biomol NMR 55:19–31

    Article  Google Scholar 

  • Hayashi K, Kojima C (2008) pCold-GST vector: a novel cold-shock vector containing GST tag for soluble protein production. Protein Expres Purif 62:120–127

    Article  Google Scholar 

  • Hiroaki H (2013) Recent applications of isotopic labeling for protein NMR in drug discovery. Expert Opin Drug Discov 8:523–536

    Article  Google Scholar 

  • Hitomi K, Kojima S, Fesus L (2015) Transglutaminases. Springer, Tokyo

    Google Scholar 

  • Jentoft N, Dearborn DG (1983) Protein labeling by reductive alkylation. Methods Enzymol 91:570–579

    Article  Google Scholar 

  • Kabsch W, Sander C (1983) Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22:2577–2637

    Article  Google Scholar 

  • Kashiwagi T, Yokoyama K-I, Ishikawa K, Ono K, Ejima D, Matsui H, Suzuki E-I (2002) Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. J Biol Chem 277:44252–44260

    Article  Google Scholar 

  • Kim TH, Mehrabi P, Ren Z, Sljoka A, Ing C, Bezginov A, Ye L, Pomès R, Prosser RS, Pai EF (2017) The role of dimer asymmetry and protomer dynamics in enzyme catalysis. Science 355:eaag2355

    Article  Google Scholar 

  • Kitevski-LeBlanc JL, Prosser RS (2012) Current applications of 19F NMR to studies of protein structure and dynamics. Prog Nucl Magn Reson Spectrosc 62:1–33

    Article  Google Scholar 

  • Larda ST, Bokoch MP, Evanics F, Prosser RS (2012) Lysine methylation strategies for characterizing protein conformations by NMR. J Biomol NMR 54:199–209

    Article  Google Scholar 

  • Leung EWW, Yagi H, Harjani JR, Mulcair MD, Scanlon MJ, Baell JB, Norton RS (2014) 19F NMR as a probe of ligand interactions with the iNOS binding site of SPRY domain-containing SOCS box protein 2. Chem Biol Drug Des 84:616–625

    Article  Google Scholar 

  • Li Y, Han J, Zhang Y, Cao F, Liu Z, Li S, Wu J, Hu C, Wang Y, Shuai J, Chen J, Cao L, Li D, Shi P, Tian C, Zhang J, Dou Y, Li G, Chen Y, Lei M (2016) Structural basis for activity regulation of MLL family methyltransferases. Nature 530:447–452

    Article  ADS  Google Scholar 

  • Liu JJ, Horst R, Katritch V, Stevens RC, Wüthrich K (2012) Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335:1106–1110

    Article  ADS  Google Scholar 

  • Manglik A, Kim TH, Masureel M, Altenbach C, Yang Z, Hilger D, Lerch MT, Kobilka TS, Thian FS, Hubbell WL, Prosser RS, Kobilka BK (2015) Structural insights into the dynamic process of β2-Adrenergic receptor signaling. Cell 161:1101–1111

    Article  Google Scholar 

  • Mishra NK, Urick AK, Ember, S. W. J., Schönbrunn E, Pomerantz WC (2014) Fluorinated aromatic amino acids are sensitive 19F NMR probes for bromodomain-ligand interactions. ACS Chem Biol 9:2755–2760

    Article  Google Scholar 

  • Norton R, Leung E, Chandrashekaran I, MacRaild C (2016) Applications of 19F-NMR in fragment-based drug discovery. Molecules 21:860

    Article  Google Scholar 

  • Ohtsuka T, Sawa A, Kawabata R, Nio N, Motoki M (2000a) Substrate specificities of microbial transglutaminase for primary amines. J Agric Food Chem 48:6230–6233

    Article  Google Scholar 

  • Ohtsuka T, Ota M, Nio N, Motoki M (2000b) Comparison of substrate specificities of transglutaminases using synthetic peptides as acyl donors. Biosci Biotechnol Biochem 64:2608–2613

    Article  Google Scholar 

  • Religa TL, Ruschak AM, Rosenzweig R, Kay LE (2011) Site-directed methyl group Labeling as an NMR probe of structure and dynamics in supramolecular protein systems: applications to the proteasome and to the ClpP protease. J Am Chem Soc 133:9063–9068

    Article  Google Scholar 

  • Rule GS, Pratt EA, Simplaceanu V, Ho C (1987) Nuclear magnetic resonance and molecular genetic studies of the membrane-bound d-lactate dehydrogenase of Escherichia coli. Biochemistry 26:549–556

    Article  Google Scholar 

  • Sato H, Hayashi E, Yamda N, Yatagai M, Takahara Y (2001) Further studies on the site-specific protein modification by microbial transglutaminase. Bioconjug Chem 12:701–710

    Article  Google Scholar 

  • Sattler M (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog Nucl Magn Reson Spectrosc 34:93–158

    Article  Google Scholar 

  • Shimba N, Yamada N, Yokoyama K-I, Suzuki E-I (2002a) Enzymatic labeling of arbitrary proteins. Anal Biochem 301:123–127

    Article  Google Scholar 

  • Shimba N, Yokoyama K-I, Suzuki E-I (2002b) NMR-based screening method for transglutaminases: rapid analysis of their substrate specificities and reaction rates. J Agric Food Chem 50:1330–1334

    Article  Google Scholar 

  • Spolaore B, Raboni S, Ramos Molina A, Satwekar A, Damiano N, Fontana A (2012) Local unfolding is required for the site-specific protein modification by transglutaminase. Biochemistry 51:8679–8689

    Article  Google Scholar 

  • Staus DP, Strachan RT, Manglik A, Pani B, Kahsai AW, Kim TH, Wingler LM, Ahn S, Chatterjee A, Masoudi A, Kruse AC, Pardon E, Steyaert J, Weis WI, Prosser RS, Kobilka BK, Costa T, Lefkowitz RJ (2016) Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature 535:448–452

    Article  ADS  Google Scholar 

  • Strop P (2014) Versatility of microbial transglutaminase. Bioconjug Chem 25:855–862

    Article  Google Scholar 

  • Tagami U, Shimba N, Nakamura M, Yokoyama K-I, Suzuki E-I, Hirokawa T (2009) Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis. Protein Eng Des Sel 22:747–752

    Article  Google Scholar 

  • Taoka K-I, Ohki I, Tsuji H, Furuita K, Hayashi K, Yanase T, Yamaguchi M, Nakashima C, Purwestri YA, Tamaki S, Ogaki Y, Shimada C, Nakagawa A, Kojima C, Shimamoto K (2011) 14-3-3 proteins act as intracellular receptors for rice Hd3a florigen. Nature 476:332–335

    Article  ADS  Google Scholar 

  • Wang GF, Li C, Pielak GJ (2010a) 19F NMR studies of α-synuclein-membrane interactions. Protein Sci 19:1686–1691

    Article  Google Scholar 

  • Wang GF, Li C, Pielak GJ (2010b) Probing the micelle-bound aggregation-prone state of α-synuclein with 19F NMR spectroscopy. ChemBioChem 11:1993–1996

    Article  Google Scholar 

  • Willard L, Ranjan A, Zhang H, Monzavi H, Boyko RF, Sykes BD, Wishart DS (2003) VADAR: a web server for quantitative evaluation of protein structure quality. Nucl Acids Res 31:3316–3319

    Article  Google Scholar 

  • Xie Q, Fulton DB, Andreotti AH (2014) A selective NMR probe to monitor the conformational transition from inactive to active kinase. ACS Chem Biol 10:262–268

    Article  Google Scholar 

  • Xu RX, Nettesheim D, Olejniczak ET, Meadows R, Gemmecker G, Fesik SW (1993) 1H, 13C, and 15N assignments and secondary structure of the FK506 binding protein when bound to ascomycin. Biopolymers 33:535–550

    Article  Google Scholar 

  • Ye L, Larda ST, Li YFF, Manglik A, Prosser RS (2015) A comparison of chemical shift sensitivity of trifluoromethyl tags: optimizing resolution in 19F NMR studies of proteins. J Biomol NMR 62:97–103

    Article  Google Scholar 

  • Ye L, Van Eps N, Zimmer M, Ernst OP, Prosser RS (2016) Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533:265–268

    Article  ADS  Google Scholar 

  • Yokoyama K, Nio N, Kikuchi Y (2004) Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol 64:447–454

    Article  Google Scholar 

  • Yokoyama K, Utsumi H, Nakamura T, Ogaya D, Shimba N, Suzuki E, Taguchi S (2010) Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis. Appl Microbiol Biotechnol 87:2087–2096

    Article  Google Scholar 

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Acknowledgements

We thank M. Yoneyama for the plasmid constructions and protein purification of GF14b mutants. This work was supported in part by KAKENHI, PDIS, and AMED-CREST from JST, MEXT and AMED. Funding was provided by Ministry of Education, Culture, Sports, Science and Technology; Japan Agency for Medical Research and Development; and Ministry of Education, Culture, Sports, Science and Technology.

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Author notes

  1. Yoshikazu Hattori and David Heidenreich have contributed equally to this work.

Authors and Affiliations

  1. Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka, 565-0871, Japan

    Yoshikazu Hattori, David Heidenreich, Yuki Ono, Toshihiko Sugiki, Toshimichi Fujiwara & Chojiro Kojima

  2. Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Nishihamaboji, 180, Yamashiro-cho, Tokushima, 770-8514, Japan

    Yoshikazu Hattori

  3. Institute for Pharmaceutical Chemistry and Buchmann Institute for Life Sciences, Johann Wolfgang Goethe-University, Max-von-Laue-Strasse 9, 60438, Frankfurt am Main, Germany

    David Heidenreich

  4. Institute for Innovation Ajinomoto Co., Inc, Suzuki-cho 1-1, Kawasaki-ku, Kawasaki, 210-8681, Japan

    Kei-ichi Yokoyama

  5. Graduate School of Engineering, Yokohama National University, Tokiwadai 79-5, Hodogaya-ku, Yokohama, 240-8501, Japan

    Ei-ichiro Suzuki & Chojiro Kojima

Authors
  1. Yoshikazu Hattori
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  2. David Heidenreich
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  8. Chojiro Kojima
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Correspondence to Chojiro Kojima.

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Hattori, Y., Heidenreich, D., Ono, Y. et al. Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions. J Biomol NMR 68, 271–279 (2017). https://doi.org/10.1007/s10858-017-0125-6

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  • DOI: https://doi.org/10.1007/s10858-017-0125-6

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