Abstract
Metal binding to sites engineered in proteins can provide an increase in their stability and facilitate new functions. Besides the sites introduced in purpose, sometimes they are present accidentally as a consequence of the expression system used to produce the protein. This happens with the copper- and nickel-binding (ATCUN) motif generated by the amino-terminal residues Gly-Ser-His. This ATCUN motif is fortuitously present in many proteins, but how it affects the structural and biophysical characterization of the proteins has not been studied. In this work, we have compared the structure and biophysical properties of a small modular domain, the SH3 domain of the c-Src tyrosine kinase, cloned with and without an ATCUN motif at the N terminus. At pH 7.0, the SH3 domain with the ATCUN motif binds nickel with a binding constant Ka = 28.0 ± 3.0 mM−1. The formation of the nickel complex increases the thermal and chemical stability of the SH3 domain. A comparison of the crystal structures of the SH3 domain with and without the ATCUN motif shows that the binding of nickel does not affect the overall structure of the SH3 domain. In all crystal structures analyzed, residues Gly-Ser-His in complex with Ni2+ show a square planar geometry. The CD visible spectrum of the nickel complex shows that this geometry is also present in the solution. Therefore, our results not only show that the ATCUN motif might influence the biophysical properties of the protein, but also points to an advantageous stabilization of the protein with potential biotechnological applications.
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Abbreviations
- CD:
-
Circular dichroism
- DLS:
-
Dynamic light scattering
- Rh :
-
Hydrodynamic radius
- MW:
-
Molecular weight
- GdnHCl:
-
Guanidine hydrochloride
- PDB:
-
Protein Data Bank
References
Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res 28(1):235–242. https://doi.org/10.1093/nar/28.1.235
Peters T Jr (1960) Interaction of one mole of copper with the alpha amino group of bovine serum albumin. Biochem Biophys Acta 39:546–547. https://doi.org/10.1016/0006-3002(60)90215-8
Sankararamakrishnan R, Verma S, Kumar S (2005) ATCUN-like metal-binding motifs in proteins: identification and characterization by crystal structure and sequence analysis. Proteins 58(1):211–221. https://doi.org/10.1002/prot.20265
Alexander JL, Thompson Z, Cowan JA (2018) Antimicrobial metallopeptides. ACS Chem Biol 13(4):844–853. https://doi.org/10.1021/acschembio.7b00989
Gokhale NH, Cowan JA (2005) Inactivation of human angiotensin converting enzyme by copper peptide complexes containing ATCUN motifs. Chem Commun 47:5916–5918. https://doi.org/10.1039/b511081e
Choi YA, Keem JO, Kim CY, Yoon HR, Heo WD, Chung BH, Jung Y (2015) A novel copper-chelating strategy for fluorescent proteins to image dynamic copper fluctuations on live cell surfaces. Chem Sci 6(2):1301–1307. https://doi.org/10.1039/c4sc03027c
Sequeira AF, Turchetto J, Saez NJ, Peysson F, Ramond L, Duhoo Y, Blémont M, Fernandes VO, Gama LT, Ferreira LMA, Guerreiro CIPI, Gilles N, Darbon H, Fontes CMGA, Vincentelli R (2017) Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli. Microb Cell Fact 16(1):4. https://doi.org/10.1186/s12934-016-0618-0
Bacarizo J, Martinez-Rodriguez S, Martin-Garcia JM, Andujar-Sanchez M, Ortiz-Salmeron E, Neira JL, Camara-Artigas A (2014) Electrostatic effects in the folding of the SH3 domain of the c-Src tyrosine kinase: pH-dependence in 3D-domain swapping and amyloid formation. PLoS ONE 9(12):e113224. https://doi.org/10.1371/journal.pone.0113224
Glover SD, Tommos C (2019) A quick and colorful method to measure low-level contaminations of paramagnetic Ni(2+) in protein samples purified by immobilized metal ion affinity chromatography. Methods Enzymol 614:87–106. https://doi.org/10.1016/bs.mie.2018.08.037
Gasteiger E, Hoogland C, Gattiker A, Se D, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, pp 571–607. https://doi.org/10.1385/1-59259-890-0
Trevino SR, Scholtz JM, Pace CN (2008) Measuring and increasing protein solubility. J Pharm Sci 97(10):4155–4166. https://doi.org/10.1002/jps.21327
Pace CN, Laurents DV (1989) A new method for determining the heat capacity change for protein folding. Biochemistry 28(6):2520–2525. https://doi.org/10.1021/bi00432a026
Camara-Artigas A, Martin-Garcia JM, Morel B, Ruiz-Sanz J, Luque I (2009) Intertwined dimeric structure for the SH3 domain of the c-Src tyrosine kinase induced by polyethylene glycol binding. FEBS Lett 583(4):749–753. https://doi.org/10.1016/j.febslet.2009.01.036
Juanhuix J, Gil-Ortiz F, Cuni G, Colldelram C, Nicolas J, Lidon J, Boter E, Ruget C, Ferrer S, Benach J (2014) Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the ALBA synchrotron. J Synchrotron Radiat 21(Pt 4):679–689. https://doi.org/10.1107/S160057751400825X
Kabsch W (2010) XDS. Acta crystallographica Section D, Biological crystallography 66 (Pt 2):125–132. doi:10.1107/S0907444909047337
Vonrhein C, Flensburg C, Keller P, Sharff A, Smart O, Paciorek W, Womack T, Bricogne G (2011) Data processing and analysis with the autoPROC toolbox. Acta Crystallogr D Biol Crystallogr 67(Pt 4):293–302. https://doi.org/10.1107/S0907444911007773
Evans PR (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr 67(Pt 4):282–292. https://doi.org/10.1107/S090744491003982X
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AG, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67(Pt 4):235–242. https://doi.org/10.1107/S0907444910045749
Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC, Richardson JS, Terwilliger TC, Zwart PH (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. https://doi.org/10.1107/S0907444909052925
Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta crystallographica Section D, Biological crystallography 68 (Pt 4):352–367. doi:10.1107/S0907444912001308
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1):2126–2132. https://doi.org/10.1107/S0907444904019158
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501. https://doi.org/10.1107/S0907444910007493
Chen VB, Arendall WB 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66(Pt 1):12–21. https://doi.org/10.1107/S0907444909042073
Joosten RP, Long F, Murshudov GN, Perrakis A (2014) The PDB_REDO server for macromolecular structure model optimization. IUCrJ 1(Pt 4):213–220. https://doi.org/10.1107/S2052252514009324
Stanyon HF, Cong X, Chen Y, Shahidullah N, Rossetti G, Dreyer J, Papamokos G, Carloni P, Viles JH (2014) Developing predictive rules for coordination geometry from visible circular dichroism of copper(II) and nickel(II) ions in histidine and amide main-chain complexes. FEBS J 281(17):3945–3954. https://doi.org/10.1111/febs.12934
Olsson MH, Sondergaard CR, Rostkowski M, Jensen JH (2011) PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J Chem Theory Comput 7(2):525–537. https://doi.org/10.1021/ct100578z
Dunbar RC, Martens J, Berden G, Oomens J (2018) Binding of divalent metal ions with deprotonated peptides: do gas-phase anions parallel the condensed phase? J Phys Chem A 122(25):5589–5596. https://doi.org/10.1021/acs.jpca.8b02926
Bacarizo J, Camara-Artigas A (2013) Atomic resolution structures of the c-Src SH3 domain in complex with two high-affinity peptides from classes I and II. Acta Crystallogr D Biol Crystallogr 69(Pt 5):756–766. https://doi.org/10.1107/S0907444913001522
Janiak C (2000) A critical account on π–π stacking in metal complexes with aromatic nitrogen-containing ligands. J Chem Soc Dalton Trans 21:3885–3896. https://doi.org/10.1039/B003010O
Laussac JP, Sarkar B (1984) Characterization of the copper(II)- and nickel(II)-transport site of human serum albumin. Studies of copper(II) and nickel(II) binding to peptide 1–24 of human serum albumin by 13C and 1H NMR spectroscopy. Biochemistry 23 (12):2832–2838. doi:10.1021/bi00307a046
Harford C, Sarkar B (1995) Neuromedin C binds Cu(II) and Ni(II) via the ATCUN motif: implications for the CNS and cancer growth. Biochem Biophys Res Commun 209(3):877–882. https://doi.org/10.1006/bbrc.1995.1580
Neupane KP, Aldous AR, Kritzer JA (2013) Macrocyclization of the ATCUN motif controls metal binding and catalysis. Inorg Chem 52(5):2729–2735. https://doi.org/10.1021/ic302820z
Yaniv O, Halfon Y, Shimon LJ, Bayer EA, Lamed R, Frolow F (2012) Structure of CBM3b of the major cellulosomal scaffoldin subunit ScaA from Acetivibrio cellulolyticus. Acta Crystallogr Sect F Struct Biol Cryst Commun 68(Pt 1):8–13. https://doi.org/10.1107/S174430911104807X
Athanasiadis A, Placido D, Maas S, Brown BA 2nd, Lowenhaupt K, Rich A (2005) The crystal structure of the Zbeta domain of the RNA-editing enzyme ADAR1 reveals distinct conserved surfaces among Z-domains. J Mol Biol 351(3):496–507. https://doi.org/10.1016/j.jmb.2005.06.028
Pichlo C, Juetten L, Wojtalla F, Schacherl M, Diaz D, Baumann U (2019) Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1. J Biol Chem 294(30):11525–11535. https://doi.org/10.1074/jbc.RA119.009029
Higgins MA, Suits MD, Marsters C, Boraston AB (2014) Structural and functional analysis of fucose-processing enzymes from Streptococcus pneumoniae. J Mol Biol 426(7):1469–1482. https://doi.org/10.1016/j.jmb.2013.12.006
Zhao J, Du Y, Horton JR, Upadhyay AK, Lou B, Bai Y, Zhang X, Du L, Li M, Wang B, Zhang L, Barbieri JT, Khuri FR, Cheng X, Fu H (2011) Discovery and structural characterization of a small molecule 14–3-3 protein-protein interaction inhibitor. Proc Natl Acad Sci USA 108(39):16212–16216. https://doi.org/10.1073/pnas.1100012108
Chaudhuri BN, Lange SC, Myers RS, Davisson VJ, Smith JL (2003) Toward understanding the mechanism of the complex cyclization reaction catalyzed by imidazole glycerolphosphate synthase: crystal structures of a ternary complex and the free enzyme. Biochemistry 42(23):7003–7012. https://doi.org/10.1021/bi034320h
Chaudhuri BN, Lange SC, Myers RS, Chittur SV, Davisson VJ, Smith JL (2001) Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (beta/alpha)8 barrel joins two active sites. Structure (London, England: 1993) 9(10):987–997
Donaldson LW, Skrynnikov NR, Choy WY, Muhandiram DR, Sarkar B, Forman-Kay JD, Kay LE (2001) Structural characterization of proteins with an attached ATCUN motif by paramagnetic relaxation enhancement NMR spectroscopy. J Am Chem Soc 123(40):9843–9847. https://doi.org/10.1021/ja011241p
Marsh JA, Neale C, Jack FE, Choy WY, Lee AY, Crowhurst KA, Forman-Kay JD (2007) Improved structural characterizations of the drkN SH3 domain unfolded state suggest a compact ensemble with native-like and non-native structure. J Mol Biol 367(5):1494–1510. https://doi.org/10.1016/j.jmb.2007.01.038
Arnold FH, Zhang JH (1994) Metal-mediated protein stabilization. Trends Biotechnol 12(5):189–192. https://doi.org/10.1016/0167-7799(94)90081-7
Yu Y, Zhou M, Kirsch F, Xu C, Zhang L, Wang Y, Jiang Z, Wang N, Li J, Eitinger T, Yang M (2014) Planar substrate-binding site dictates the specificity of ECF-type nickel/cobalt transporters. Cell Res 24(3):267–277. https://doi.org/10.1038/cr.2013.172
Neupane KP, Aldous AR, Kritzer JA (2014) Metal-binding and redox properties of substituted linear and cyclic ATCUN motifs. J Inorg Biochem 139:65–76. https://doi.org/10.1016/j.jinorgbio.2014.06.004
Mital M, Zawisza IA, Wiloch MZ, Wawrzyniak UE, Kenche V, Wroblewski W, Bal W, Drew SC (2016) Copper exchange and redox activity of a prototypical 8-hydroxyquinoline: implications for therapeutic chelation. Inorg Chem 55(15):7317–7319. https://doi.org/10.1021/acs.inorgchem.6b00832
Alexander JL, Thompson Z, Yu Z, Cowan JA (2019) Cu-ATCUN derivatives of sub5 exhibit enhanced antimicrobial activity via multiple modes of action. ACS Chem Biol 14(3):449–458. https://doi.org/10.1021/acschembio.8b01087
Acknowledgements
This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (Spain) and FEDER (EU) [BIO2016-78020-R and BIO2016-78746-C2-1-R]. Data collection was supported by ALBA (Barcelona, Spain) [BAG 2018072903].
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Plaza-Garrido, M., Salinas-García, M.C., Martínez, J.C. et al. The effect of an engineered ATCUN motif on the structure and biophysical properties of the SH3 domain of c-Src tyrosine kinase. J Biol Inorg Chem 25, 621–634 (2020). https://doi.org/10.1007/s00775-020-01785-0
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DOI: https://doi.org/10.1007/s00775-020-01785-0