Structure and dynamics of Helicobacter pylori nickel-chaperone HypA: an integrated approach using NMR spectroscopy, functional assays and computational tools

  • Chris A. E. M. Spronk
  • Szymon Żerko
  • Michał Górka
  • Wiktor Koźmiński
  • Benjamin Bardiaux
  • Barbara Zambelli
  • Francesco Musiani
  • Mario Piccioli
  • Priyanka Basak
  • Faith C. Blum
  • Ryan C. Johnson
  • Heidi Hu
  • D. Scott Merrell
  • Michael Maroney
  • Stefano Ciurli
Original Paper


Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3–7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.


Metallochaperones Metal transport Molecular dynamics Nuclear magnetic resonance Computational chemistry Nickel 



This work was supported by a grant from the Polish National Science Centre (MAESTRO—2015/18/A/ST4/00270 to MG, SZ, WK), by a grant from the U.S. National Institutes of Health (NIH—R01-GM069696 to MJM), by the Institut Pasteur, CNRS and the French Institute of Bioinformatics (IFB; ANR-11-INBS-0013, to BB), by the European Cooperation in Science and Technology (COST) Action 15133 (MP), and by the Department of Pharmacy and Biotechnology of the University of Bologna (SC, BZ, FM). The NMR experiments were partially obtained in the frames of access to NMR infrastructure by EuroBioNMR EEIG ( The Center for Magnetic Resonance of the University of Florence (CERM) provided access to the high-field NMR spectrometers, and Fabio Calogiuri is acknowledged for spectra data collection.

Supplementary material

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Supplementary material 1 (DOCX 2135 kb)


  1. 1.
    Testerman TL, Morris J (2014) World J Gastroenterol 20:12781–12808CrossRefPubMedGoogle Scholar
  2. 2.
    Gobert AP, Wilson KT (2016) Trends Microbiol 24:366–376CrossRefPubMedGoogle Scholar
  3. 3.
    Eusebi LH, Zagari RM, Bazzoli F (2014) Helicobacter 19(Suppl 1):1–5CrossRefPubMedGoogle Scholar
  4. 4.
    IARC helicobacter pylori Working Group (2014) Helicobacter pylori eradication as a strategy for preventing gastric cancer. International Agency for Research on Cancer (IARC Working Group Reports, No. 8). Lyon, France. Available from:
  5. 5.
    Stathis A, Bertoni F, Zucca E (2010) Expert Opin Pharmacother 11:2141–2152CrossRefPubMedGoogle Scholar
  6. 6.
    World health organization (WHO) (2017) Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibioticsGoogle Scholar
  7. 7.
    Zambelli B, Ciurli S (2013) Met Ions Life Sci 13:321–357CrossRefPubMedGoogle Scholar
  8. 8.
    Zambelli B, Musiani F, Benini S, Ciurli S (2011) Acc Chem Res 44:520–530CrossRefPubMedGoogle Scholar
  9. 9.
    Kusters JG, van Vliet AH, Kuipers EJ (2006) Clin Microbiol Rev 19:449–490CrossRefPubMedGoogle Scholar
  10. 10.
    Yamaoka Y (2010) Nat Rev Gastro Hepat 7:629–641CrossRefGoogle Scholar
  11. 11.
    Bauerfeind P, Garner R, Dunn BE, Mobley HLT (1997) Gut 40:25–30CrossRefPubMedGoogle Scholar
  12. 12.
    Maroney MJ, Ciurli S (2014) Chem Rev 114:4206–4228CrossRefGoogle Scholar
  13. 13.
    Eaton KA, Brooks CL, Morgan DR, Krakowka S (1991) Infect Immun 59:2470–2475PubMedCentralPubMedGoogle Scholar
  14. 14.
    Eaton KA, Krakowka S (1994) Infect Immun 62:3604–3607PubMedCentralPubMedGoogle Scholar
  15. 15.
    Campanale M, Nucera E, Ojetti V, Cesario V, Di Rienzo TA, D’Angelo G, Pecere S, Barbaro F, Gigante G, De Pasquale T, Rizzi A, Cammarota G, Schiavino D, Franceschi F, Gasbarrini A (2014) Dig Dis Sci 59:1851–1855CrossRefGoogle Scholar
  16. 16.
    Mazzei L, Musiani F, Ciurli S (2017) In: Zamble D, Rowińska-Żyrek M, Kozłowski H (eds) The biological chemistry of nickel. Royal Society of Chemistry, pp 60–97Google Scholar
  17. 17.
    Pedroso MM, Ely F, Carpenter MC, Mitić NN, Gahan LR, Ollis DL, Wilcox DE, Schenk GG (2017) Biochemistry 56:3328–3336CrossRefGoogle Scholar
  18. 18.
    Fong YH, Wong HC, Yuen MH, Lau PH, Chen YW, Wong KB (2013) PLoS Biol 11:e1001678CrossRefPubMedGoogle Scholar
  19. 19.
    Farrugia MA, Macomber L, Hausinger RP (2013) J Biol Chem 288:13178–13185CrossRefPubMedGoogle Scholar
  20. 20.
    Banaszak K, Martin-Diaconescu V, Bellucci M, Zambelli B, Rypniewski W, Maroney MJ, Ciurli S (2012) Biochem J 441:1017–1026CrossRefPubMedGoogle Scholar
  21. 21.
    Mehta N, Olson JW, Maier RJ (2003) J Bacteriol 185:726–734CrossRefPubMedGoogle Scholar
  22. 22.
    Casalot L, Rousset M (2001) Trends Microbiol 9:228–237CrossRefGoogle Scholar
  23. 23.
    Blum FC, Hu HQ, Servetas SL, Benoit SL, Maier RJ, Maroney MJ, Merrell DS (2017) PLoS One 12:e0183260CrossRefPubMedGoogle Scholar
  24. 24.
    Hu HQ, Huang HT, Maroney MJ (2018) Biochemistry 57:2932–2942CrossRefGoogle Scholar
  25. 25.
    Yang X, Li H, Cheng T, Xia W, Lai YT, Sun H (2014) Metallomics 6:1731–1736CrossRefGoogle Scholar
  26. 26.
    Olson JW, Mehta NS, Maier RJ (2001) Mol Microbiol 39:176–182CrossRefGoogle Scholar
  27. 27.
    Hu HQ, Johnson RC, Merrell DS, Maroney MJ (2017) Biochemistry 56:1105–1116CrossRefPubMedGoogle Scholar
  28. 28.
    Johnson RC, Hu HQ, Merrell DS, Maroney MJ (2015) Metallomics 7:674–682CrossRefPubMedGoogle Scholar
  29. 29.
    Kennedy DC, Herbst RW, Iwig JS, Chivers PT, Maroney MJ (2007) J Am Chem Soc 129:16–17CrossRefPubMedGoogle Scholar
  30. 30.
    Herbst RW, Perovic I, Martin-Diaconescu V, O’Brien K, Chivers PT, Pochapsky SS, Pochapsky TC, Maroney MJ (2010) J Am Chem Soc 132:10338–10351CrossRefPubMedGoogle Scholar
  31. 31.
    Xia W, Li H, Sze K-H, Sun H (2009) J Am Chem Soc 131:10031–10040CrossRefPubMedGoogle Scholar
  32. 32.
    Watanabe S, Arai T, Matsumi R, Atomi H, Imanaka T, Miki K (2009) J Mol Biol 394:448–459CrossRefPubMedGoogle Scholar
  33. 33.
    Watanabe S, Kawashima T, Nishitani Y, Kanai T, Wada T, Inaba K, Atomi H, Imanaka T, Miki K (2015) Proc Natl Acad Sci USA 112:7701–7706CrossRefPubMedGoogle Scholar
  34. 34.
    Kwon S, Watanabe S, Nishitani Y, Kawashima T, Kanai T, Atomi H, Miki K (2018) Proc Natl Acad Sci USA 115:7045–7050CrossRefPubMedGoogle Scholar
  35. 35.
    Stingl K, De Reuse H (2005) Int J Med Microbiol 295:307–315CrossRefGoogle Scholar
  36. 36.
    Sachs G, Weeks DL, Wen Y, Marcus EA, Scott DR, Melchers K (2005) Physiology 20:429–438CrossRefGoogle Scholar
  37. 37.
    Scott DR, Marcus EA, Weeks DL, Sachs G (2002) Gastroenterology 123:187–195CrossRefGoogle Scholar
  38. 38.
    Wen Y, Marcus EA, Matrubutham U, Gleeson MA, Scott DR, Sachs G (2003) Infect Immun 71:5921–5939CrossRefPubMedGoogle Scholar
  39. 39.
    Jones MD, Li Y, Zamble DB (2018) Proc Nat Acad Sci USA 115(36):8966–8971CrossRefPubMedGoogle Scholar
  40. 40.
    Stola M, Musiani F, Mangani S, Turano P, Safarov N, Zambelli B, Ciurli S (2006) Biochemistry 45:6495–6509CrossRefGoogle Scholar
  41. 41.
    Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) J Biomol NMR 6:277–293CrossRefGoogle Scholar
  42. 42.
    Keller RLJ (2004) Swiss Federal Institute of Technology, ZurichGoogle Scholar
  43. 43.
    Stanek J, Augustyniak R, Kozminski W (2012) J Magn Reson 214:91–102CrossRefPubMedGoogle Scholar
  44. 44.
    Goddard TD, Kneller DG (2000) University of California, San FranciscoGoogle Scholar
  45. 45.
    Shen Y, Delaglio F, Cornilescu G, Bax A (2009) J Biomol NMR 44:213–223CrossRefPubMedGoogle Scholar
  46. 46.
    Rieping W, Habeck M, Bardiaux B, Bernard A, Malliavin TE, Nilges M (2007) Bioinformatics 23:381–382CrossRefPubMedGoogle Scholar
  47. 47.
    Krieger E, Vriend G (2014) Bioinformatics 30:2981–2982CrossRefPubMedGoogle Scholar
  48. 48.
    Herrmann T, Güntert P, Wüthrich K (2002) J Mol Biol 319:209–227CrossRefPubMedGoogle Scholar
  49. 49.
    Nilges M, Bernard A, Bardiaux B, Malliavin TE, Habeck M, Rieping W (2008) Structure 16:1305–1312CrossRefPubMedGoogle Scholar
  50. 50.
    Mareuil F, Malliavin TE, Nilges M, Bardiaux B (2015) J Biomol NMR 62:425–438CrossRefPubMedGoogle Scholar
  51. 51.
    Krieger E, Joo K, Lee J, Lee J, Raman S, Thompson J, Tyka M, Baker D, Karplus K (2009) Proteins 77:114–122CrossRefPubMedGoogle Scholar
  52. 52.
    Darden T, York D, Pedersen L (1993) J Chem Phys 98:10089–10092CrossRefGoogle Scholar
  53. 53.
    Vriend G (1990) J Mol Graph 8:52–56CrossRefGoogle Scholar
  54. 54.
    Koradi R, Billeter M, Wuthrich K (1996) J Mol Graph 14:51–55CrossRefGoogle Scholar
  55. 55.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) J Comput Chem 25:1605–1612CrossRefGoogle Scholar
  56. 56.
    Gordon JC, Myers JB, Folta T, Shoja V, Heath LS, Onufriev A (2005) Nucleic Acids Res 33:W368–W371CrossRefPubMedGoogle Scholar
  57. 57.
    Myers J, Grothaus G, Narayanan S, Onufriev A (2006) Proteins 63:928–938CrossRefGoogle Scholar
  58. 58.
    Anandakrishnan R, Aguilar B, Onufriev AV (2012) Nucleic Acids Res 40:W537–W541CrossRefPubMedGoogle Scholar
  59. 59.
    Hornak V, Abel R, Okur A, Strockbine B, Roitberg A, Simmerling C (2006) Proteins 65:712–725CrossRefPubMedGoogle Scholar
  60. 60.
    Jorgensen WL, Chandrasekhar L, Madura JD, Impey RW, Klein ML (1983) J Chem Phys 79:926–935CrossRefGoogle Scholar
  61. 61.
    Peters MB, Yang Y, Wang B, Fusti-Molnar L, Weaver MN, Merz KM Jr (2010) J Chem Theory Comput 6:2935–2947CrossRefPubMedGoogle Scholar
  62. 62.
    Berendsen HJC, van der Spoel D, van Drunen R (1995) Comput Phys Commun 91:43–56CrossRefGoogle Scholar
  63. 63.
    Lindahl E, Hess B, van der Spoel D (2001) J Mol Model 7:306–317CrossRefGoogle Scholar
  64. 64.
    Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC (2005) J Comput Chem 26:1701–1718CrossRefGoogle Scholar
  65. 65.
    Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) J Chem Phys 81:3684–3690CrossRefGoogle Scholar
  66. 66.
    Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) J Chem Phys 103:8577–8593CrossRefGoogle Scholar
  67. 67.
    Hoover WG (1985) Phys Rev A 31:1695–1697CrossRefGoogle Scholar
  68. 68.
    Nosé S (2002) Mol Phys 100:191–198CrossRefGoogle Scholar
  69. 69.
    Nosé S, Klein ML (1983) Mol Phys 50:1055–1076CrossRefGoogle Scholar
  70. 70.
    Parrinello M, Rahman A (1981) J Appl Phys 52:7182–7190CrossRefGoogle Scholar
  71. 71.
    Daura X, Gademann K, Jaun B, Seebach D, van Gunsteren WF, Mark AE (1999) Angew Chem 38:236–240CrossRefGoogle Scholar
  72. 72.
    Horton RM, Ho SN, Pullen JK, Hunt HD, Cai Z, Pease LR (1993) Methods Enzymol 217:270–279CrossRefPubMedGoogle Scholar
  73. 73.
    Williamson MP (2013) Prog Nucl Magn Reson Spectrosc 73:1–16CrossRefPubMedGoogle Scholar
  74. 74.
    Piccioli M, Turano P (2015) Coord Chem Rev 284:313–328CrossRefGoogle Scholar
  75. 75.
    Banci L, Bertini I, Luchinat C, Piccioli M, Scozzafava A, Turano P (1989) Inorg Chem 28:4650–4656CrossRefGoogle Scholar
  76. 76.
    Bertini I, Capozzi F, Ciurli S, Luchinat C, Messori L, Piccioli M (1992) J Am Chem Soc 114:3332–3340CrossRefGoogle Scholar
  77. 77.
    Neese F (2012) Wiley Interdiscip Rev Comput Mol Sci 2:73–78CrossRefGoogle Scholar
  78. 78.
    Becke AD (1993) J Chem Phys 98:1372–1377CrossRefGoogle Scholar
  79. 79.
    Lee C, Yang W, Parr RG (1988) Phys Rev B 37:785–789CrossRefGoogle Scholar
  80. 80.
    Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery JA Jr, Peralta JE, Ogliaro F, Bearpark MJ, Heyd J, Brothers EN, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell AP, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam NJ, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas Ö, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ (2009) Gaussian Inc., WallingfordGoogle Scholar
  81. 81.
    Frisch MJ, Pople JA (1984) J Chem Phys 80:3265CrossRefGoogle Scholar
  82. 82.
    Hay PJ, Wadt WR (1985) J Chem Phys 82:299–310CrossRefGoogle Scholar
  83. 83.
    Ponzoni L, Polles G, Carnevale V, Micheletti C (2015) Structure 23:1516–1525CrossRefGoogle Scholar
  84. 84.
    Sharma D, Rajarathnam K (2000) J Biomol NMR 18:165–171CrossRefGoogle Scholar
  85. 85.
    Allegrozzi M, Bertini I, Janik MBL, Lee Y-M, Liu G, Luchinat C (2000) J Am Chem Soc 122:4154–4161CrossRefGoogle Scholar
  86. 86.
    Banci L, Piccioli M (1996) Encyclopedia of magnetic resonance. pp 1365–1378Google Scholar
  87. 87.
    Ming LJ, Banci L, Luchinat C, Bertini I, Valentine JS (1988) Inorg Chem 27:4458–4463CrossRefGoogle Scholar
  88. 88.
    Bertini I, Donaire A, Monnanni R, Moratal J-M, Salgado J (1992) J Chem Soc Dalton Trans. CrossRefGoogle Scholar
  89. 89.
    Donaire A, Salgado J, Moratal JM (1998) Biochemistry 37:8659–8673CrossRefPubMedGoogle Scholar
  90. 90.
    Rossi P, Swapna GVT, Huang YJ, Aramini JM, Anklin C, Conover K, Hamilton K, Xiao R, Acton T, Ertekin A, Everett JK, Montelione GT (2010) J Biomol NMR 46:11–22CrossRefGoogle Scholar
  91. 91.
    Bertini I, Luchinat C (1986) NMR of paramagnetic molecules in biological systems. Benjamin-Cummings, Menlo ParkGoogle Scholar

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© SBIC 2018

Authors and Affiliations

  1. 1.JSC SpronkVilniusLithuania
  2. 2.Department of Molecular and Cell Biology, Leicester Institute of Structural and Chemical BiologyUniversity of LeicesterLeicesterUK
  3. 3.Faculty of Chemistry, Biological and Chemical Research CentreUniversity of WarsawWarsawPoland
  4. 4.Faculty of Physics, Division of Biophysics, Institute of Experimental PhysicsUniversity of WarsawWarsawPoland
  5. 5.Structural Bioinformatics Unit, Department of Structural Biology and ChemistryInstitut Pasteur, CNRS UMR3528ParisFrance
  6. 6.Laboratory of Bioinorganic Chemistry, Department of Pharmacy and BiotechnologyUniversity of BolognaBolognaItaly
  7. 7.Center for Magnetic Resonance, Department of ChemistryUniversity of FlorenceFlorenceItaly
  8. 8.Department of ChemistryUniversity of MassachusettsAmherstUSA
  9. 9.Department of Microbiology and ImmunologyUniformed Services University of the Health SciencesBethesdaUSA
  10. 10.Department of ChemistryUniversity of MassachusettsAmherstUSA

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