Abstract
Lanthanoid pseudo-contact shift (PCS) provides long-range structural information between a paramagnetic tag and protein nuclei. However, for proteins with native cysteines, site-specific attachment may only utilize functional groups orthogonal to sulfhydryl chemistry. Here we report two lanthanoid probes, DTTA-C3-yne and DTTA-C4-yne, which can be conjugated to an unnatural amino acid pAzF in the target protein via azide-alkyne cycloaddition. Demonstrated with ubiquitin and cysteine-containing enzyme EIIB, we show that large PCSs of distinct profiles can be generated for each tag/lanthanoid combination. The DTTA-based lanthanoid tags are associated with large magnetic susceptibility tensors owing to the rigidity of the tags. In particular, introduction of the DTTA-C3 tag affords intermolecular PCSs and enables structural characterization of a transient protein complex between ubiquitin and a UBA domain. Together, we have expanded the repertoire of paramagnetic tags and the applicability of paramagnetic NMR.
References
Bermejo GA, Clore GM, Schwieters CD (2012) Smooth statistical torsion angle potential derived from a large conformational database via adaptive kernel density estimation improves the quality of NMR protein structures. Protein Sci 21:1824–1836
Cai M, Williams DC Jr, Wang G, Lee BR, Peterkofsky A, Clore GM (2003) Solution structure of the phosphoryl transfer complex between the signal-transducing protein IIAGlucose and the cytoplasmic domain of the glucose transporter IICBGlucose of the Escherichia coli glucose phosphotransferase system. J Biol Chem 278:25191–25206
Chin JW (2014) Expanding and reprogramming the genetic code of cells and animals. Annu Rev Biochem 83:379–408
Chin JW, Santoro SW, Martin AB, King DS, Wang L, Schultz PG (2002) Addition of p-azido-l-phenylalanine to the genetic code of Escherichia coli. J Am Chem Soc 124:9026–9027
Clore GM, Venditti V (2013) Structure, dynamics and biophysics of the cytoplasmic protein-protein complexes of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Trends Biochem Sci 38:515–530
Hass MA, Liu WM, Agafonov RV, Otten R, Phung LA, Schilder JT, Kern D, Ubbink M (2015) A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy. J Biomol NMR 61:123–136
Haussinger D, Huang JR, Grzesiek S (2009) A-M8: An extremely rigid, high-affinity lanthanide chelating tag for PCS NMR spectroscopy. J Am Chem Soc 131:14761–14767
Hong V, Presolski SI, Ma C, Finn MG (2009) Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. Angew Chem Int Ed 48:9879–9883
Ikegami T, Verdier L, Sakhaii P, Grimme S, Pescatore B, Saxena K, Fiebig KM, Griesinger C (2004) Novel techniques for weak alignment of proteins in solution using chemical tags coordinating lanthanide ions. J Biomol NMR 29:339–349
Iwahara J, Tang C, Clore GM (2007) Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules. J Magn Reson 184:185–195
John M, Park AY, Pintacuda G, Dixon NE, Otting G (2005) Weak alignment of paramagnetic proteins warrants correction for residual CSA effects in measurements of pseudocontact shifts. J Am Chem Soc 127:17190–17191
Keizers PH, Saragliadis A, Hiruma Y, Overhand M, Ubbink M (2008) Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment. J Am Chem Soc 130:14802–14812
Liu Z, Tang C (2016) Ensemble structure description of Lys63-linked diubiquitin. Data Brief 7:81–88
Loh CT, Ozawa K, Tuck KL, Barlow N, Huber T, Otting G, Graham B (2013) Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins. Bioconjug Chem 24:260–268
Loh CT, Graham B, Abdelkader EH, Tuck KL, Otting G (2015) Generation of pseudocontact shifts in proteins with lanthanides using small “clickable” nitrilotriacetic acid and iminodiacetic acid tags. Chemistry 21:5084–5092
Michielssens S, Peters JH, Ban D, Pratihar S, Seeliger D, Sharma M, Giller K, Sabo TM, Becker S, Lee D, Griesinger C, de Groot BL (2014) A designed conformational shift to control protein binding specificity. Angew Chem Int Ed 53:10367–10371
Mueller TD, Feigon J (2002) Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions. J Mol Biol 319:1243–1255
Mueller TD, Kamionka M, Feigon J (2004) Specificity of the interaction between ubiquitin-associated domains and ubiquitin. J Biol Chem 279:11926–11936
Otting G (2010) Protein NMR using paramagnetic ions. Ann Rev Biophys 39:387–405
Park SH, Wang VS, Radoicic J, De Angelis AA, Berkamp S, Opella SJ (2015) Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA). J Biomol NMR 61:185–196
Pintacuda G, Park AY, Keniry MA, Dixon NE, Otting G (2006) Lanthanide labeling offers fast NMR approach to 3D structure determinations of protein-protein complexes. J Am Chem Soc 128:3696–3702
Schmitz C, Stanton-Cook MJ, Su XC, Otting G, Huber T (2008) Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts. J Biomol NMR 41:179–189
Schwieters CD, Kuszewski JJ, Clore GM (2006) Using Xplor-NIH for NMR molecular structure determination. Prog Nucl Magn Reson Spectrosc 48:47–62
Shishmarev D, Otting G (2013) How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic metal tags? A modelling study. J Biomol NMR 56:203–216
Su XC, Otting G (2010) Paramagnetic labelling of proteins and oligonucleotides for NMR. J Biomol NMR 46:101–112
Swanson KA, Hicke L, Radhakrishnan I (2006) Structural basis for monoubiquitin recognition by the Ede1 UBA domain. J Mol Biol 358:713–724
Swarbrick JD, Ung P, Chhabra S, Graham B (2011a) An iminodiacetic acid based lanthanide binding tag for paramagnetic exchange NMR spectroscopy. Angew Chem Int Ed 50:4403–4406
Swarbrick JD, Ung P, Su XC, Maleckis A, Chhabra S, Huber T, Otting G, Graham B (2011b) Engineering of a bis-chelator motif into a protein alpha-helix for rigid lanthanide binding and paramagnetic NMR spectroscopy. Chem Commun 47:7368–7370
Tang C, Clore GM (2006) A simple and reliable approach to docking protein-protein complexes from very sparse NOE-derived intermolecular distance restraints. J Biomol NMR 36:37–44
Vijay-Kumar S, Bugg CE, Cook WJ (1987) Structure of ubiquitin refined at 1.8 A resolution. J Mol Biol 194:531–544
Walinda E, Morimoto D, Sugase K, Konuma T, Tochio H, Shirakawa M (2014) Solution structure of the ubiquitin-associated (UBA) domain of human autophagy receptor NBR1 and its interaction with ubiquitin and polyubiquitin. J Biol Chem 289:13890–13902
Xing Q, Huang P, Yang J, Sun JQ, Gong Z, Dong X, Guo DC, Chen SM, Yang YH, Wang Y, Yang MH, Yi M, Ding YM, Liu ML, Zhang WP, Tang C (2014) Visualizing an ultra-weak protein–protein interaction in phosphorylation signaling. Angew Chem Int Ed 53:11501–11505
Young TS, Ahmad I, Yin JA, Schultz PG (2010) An enhanced system for unnatural amino acid mutagenesis in E. coli. J Mol Biol 395:361–374
Zhang D, Raasi S, Fushman D (2008) Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains. J Mol Biol 377:162–180
Acknowledgements
We thank the Chinese Ministry of Science and Technology (2013CB910200 and 2016YFA0501200 to C.T.), the National Natural Science Foundation of China (31225007 to C.T., and 31400644 to X.D.), and the K.C. Wong Education Foundation for support for grants support. The research of C.T. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute. The NMR structure of ubiqutin-UBA1 complex along with chemical shift values (diamagnetic, paramagnetic, intramolecular and intermolecular) has been deposited at the PDB with accession code of 5XBO.
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Wen-Xue Jiang and Xin-Hua Gu have contributed equally.
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Jiang, WX., Gu, XH., Dong, X. et al. Lanthanoid tagging via an unnatural amino acid for protein structure characterization. J Biomol NMR 67, 273–282 (2017). https://doi.org/10.1007/s10858-017-0106-9
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DOI: https://doi.org/10.1007/s10858-017-0106-9