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High-resolution crystal structures of a “half sandwich”-type Ru(II) coordination compound bound to hen egg-white lysozyme and proteinase K

Abstract

The high-resolution X-ray crystal structures of the adducts formed between the “half sandwich”-type Ru(II) coordination compound [RuII(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]+ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes.

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References

  1. 1.

    Alessio E (2011) Bioinorganic medicinal chemistry. Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim, Germany. doi: 10.1002/9783527633104

    Google Scholar 

  2. 2.

    Monro S, Colón KL, Yin H, Roque J, Konda P, Gujar S, Thummel RP, Lilge L, Cameron CG, McFarland SA (2019) Transition metal complexes and photodynamic therapy from a tumor-centered approach: challenges, opportunities, and highlights from the development of TLD1433. Chem Rev 119(2):797–828

    CAS  PubMed  Article  Google Scholar 

  3. 3.

    Rademaker-Lakhai JM, Van Den Bongard D, Pluim D, Beijnen JH, Schellens JHM (2004) A phase I and pharmacological study with imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a novel ruthenium anticancer agent. Clin Cancer Res 10(11):3717–3727

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Lentz F, Drescher A, Lindauer A, Henke M, Hilger RA, Hartinger CG, Scheulen ME, Dittrich C, Keppler BK, Jaehde U (2009) Pharmacokinetics of a novel anticancer ruthenium complex (KP1019, FFC14A) in a phase i dose-escalation study. Anticancer Drugs 20(2):97–103

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Burris HA, Bakewell S, Bendell JC, Infante J, Jones SF, Spigel DR, Weiss GJ, Ramanathan RK, Ogden A, Von Hoff D (2016) Safety and activity of IT-139, a ruthenium-based compound, in patients with advanced solid tumours: a first-in-human, open-label, dose-escalation phase i study with expansion cohort. ESMO Open 1(6):e000154

    PubMed  Article  Google Scholar 

  6. 6.

    Trondl R, Heffeter P, Kowol CR, Jakupec MA, Berger W, Keppler BK (2014) NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chem Sci 5(8):2925–2932

    CAS  Article  Google Scholar 

  7. 7.

    Murray BS, Babak MV, Hartinger CG, Dyson PJ (2016) The development of RAPTA compounds for the treatment of tumors. Coord Chem Rev 306(P1):86–114

    CAS  Article  Google Scholar 

  8. 8.

    Yan YK, Melchart M, Habtemariam A, Sadler PJ (2005) Organometallic chemistry, biology and medicine: ruthenium arene anticancer complexes. Chem Commun 38:4764–4776

    Article  CAS  Google Scholar 

  9. 9.

    Aird RE, Cummings J, Ritchie AA, Muir M, Jodrell DI, Morris RE, Chen H, Sadler PJ (2002) In vitro and in vivo activity and cross resistance profiles of novel ruthenium (II) organometallic arene complexes in human ovarian cancer. Br J Cancer 86(10):1652–1657

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Bergamo A, Masi A, Peacock AFA, Habtemariam A, Sadler PJ, Sava G (2010) In vivo tumour and metastasis reduction and in vitro effects on invasion assays of the ruthenium RM175 and osmium AFAP51 organometallics in the mammary cancer model. J Inorg Biochem 104(1):79–86

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Bergamo A, Sava G (2015) Linking the future of anticancer metal-complexes to the therapy of tumour metastases. Chem Soc Rev 44(24):8818–8835

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Hartinger CG, Zorbas-Seifried S, Jakupec MA, Kynast B, Zorbas H, Keppler BK (2006) From bench to bedside—preclinical and early clinical development of the anticancer agent indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019 or FFC14A). J Inorg Biochem 100(5–6):891–904

    CAS  PubMed  Article  Google Scholar 

  13. 13.

    Scolaro C, Bergamo A, Brescacin L, Delfino R, Cocchietto M, Laurenczy G, Geldbach TJ, Sava G, Dyson PJ (2005) In vitro and in vivo evaluation of ruthenium(II)-arene PTA complexes. J Med Chem 48(12):4161–4171

    CAS  PubMed  Article  Google Scholar 

  14. 14.

    Nowak-Sliwinska P, Van Beijnum JR, Casini A, Nazarov AA, Wagnières G, Van Den Bergh H, Dyson PJ, Griffioen AW (2011) Organometallic ruthenium(II) arene compounds with antiangiogenic activity. J Med Chem 54(11):3895–3902

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Guichard SM, Else R, Reid E, Zeitlin B, Aird R, Muir M, Dodds M, Fiebig H, Sadler PJ, Jodrell DI (2006) Anti-tumour activity in non-small cell lung cancer models and toxicity profiles for novel ruthenium(II) based organo-metallic compounds. Biochem Pharmacol 71(4):408–415

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Alessio E (2017) Thirty years of the drug candidate NAMI-A and the myths in the field of ruthenium anticancer compounds: a personal perspective. Eur J Inorg Chem 2017(12):1549–1560

    CAS  Article  Google Scholar 

  17. 17.

    Gianferrara T, Bratsos I, Alessio E (2009) A categorization of metal anticancer compounds based on their mode of action. Dalton Trans 37:7588–7598

    Article  CAS  Google Scholar 

  18. 18.

    Meier-Menches SM, Gerner C, Berger W, Hartinger CG, Keppler BK (2018) Structure-activity relationships for ruthenium and osmium anticancer agents-towards clinical development. Chem Soc Rev 47(3):909–928

    CAS  PubMed  Article  Google Scholar 

  19. 19.

    Messori L, Merlino A (2017) Protein metalation by metal-based drugs: X-ray crystallography and mass spectrometry studies. Chem Commun 53(85):11622–11633

    CAS  Article  Google Scholar 

  20. 20.

    Rilak Simović A, Masnikosa R, Bratsos I, Alessio E (2019) Chemistry and reactivity of ruthenium(II) complexes: DNA/protein binding mode and anticancer activity are related to the complex structure. Coord Chem Rev 398:113011

    Article  CAS  Google Scholar 

  21. 21.

    Aitken JB, Antony S, Weekley CM, Lai B, Spiccia L, Harris HH (2012) Distinct cellular fates for KP1019 and NAMI-A determined by X-ray fluorescence imaging of single cells. Metallomics 4(10):1051–1056

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Levina A, Aitken JB, Gwee YY, Lim ZJ, Liu M, Singharay AM, Wong PF, Lay PA (2013) Biotransformations of anticancer ruthenium(III) complexes: an X-ray absorption spectroscopic study. Chem Eur J 19(11):3609–3619

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Adhireksan Z, Davey GE, Campomanes P, Groessl M, Clavel CM, Yu H, Nazarov AA, Yeo CHF, Ang WH, Dröge P, Rothlisberger U, Dyson PJ, Davey CA (2014) Ligand substitutions between ruthenium–cymene compounds can control protein versus DNA targeting and anticancer activity. Nat Commun 5(1):3462

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  24. 24.

    Wu B, Ong MS, Groessl M, Adhireksan Z, Hartinger CG, Dyson PJ, Davey CA (2011) A ruthenium antimetastasis agent forms specific histone protein adducts in the nucleosome core. Chem Eur J 17(13):3562–3566

    CAS  PubMed  Article  Google Scholar 

  25. 25.

    Bratsos I, Birarda G, Jedner S, Zangrando E, Alessio E (2007) Half-sandwich RuII-[9]aneS3 complexes with dicarboxylate ligands: synthesis, characterization and chemical behavior. Dalton Trans 36:4048–4058

    Article  CAS  Google Scholar 

  26. 26.

    Bratsos I, Jedner S, Bergamo A, Sava G, Gianferrara T, Zangrando E, Alessio E (2008) Half-sandwich RuII-[9]aneS3 complexes structurally similar to antitumor-active organometallic piano-stool compounds: Preparation, structural characterization and in vitro cytotoxic activity. J Inorg Biochem 102(5–6):1120–1133

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Bratsos I, Mitri E, Ravalico F, Zangrando E, Gianferrara T, Bergamo A, Alessio E (2012) New half sandwich Ru(II) coordination compounds for anticancer activity. Dalton Trans 41(24):7358–7371

    CAS  PubMed  Article  Google Scholar 

  28. 28.

    Kljun J, Bratsos I, Alessio E, Psomas G, Repnik U, Butinar M, Turk B, Turel I (2013) New uses for old drugs: attempts to convert quinolone antibacterials into potential anticancer agents containing ruthenium. Inorg Chem 52(15):9039–9052

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Serli B, Zangrando E, Gianferrara T, Scolaro C, Dyson PJ, Bergamo A, Alessio E (2005) Is the aromatic fragment of piano-stool ruthenium compounds an essential feature for anticancer activity? The development of new RuII-[9]aneS3 analogues. Eur J Inorg Chem 17:3423–3434

    Article  CAS  Google Scholar 

  30. 30.

    Rilak A, Bratsos I, Zangrando E, Kljun J, Turel I, Bugarčić ŽD, Alessio E (2012) Factors that influence the antiproliferative activity of half sandwich RuII-[9]aneS3 coordination compounds: activation kinetics and interaction with guanine derivatives. Dalton Trans 41(38):11608–11618

    CAS  PubMed  Article  Google Scholar 

  31. 31.

    Merlino A (2016) Interactions between proteins and Ru compounds of medicinal interest: a structural perspective. Coord Chem Rev 326:111–134

    CAS  Article  Google Scholar 

  32. 32.

    Sullivan MP, Groessl M, Meier SM, Kingston RL, Goldstone DC, Hartinger CG (2017) The metalation of hen egg white lysozyme impacts protein stability as shown by ion mobility mass spectrometry, differential scanning calorimetry, and X-ray crystallography. Chem Commun 53(30):4246–4249

    CAS  Article  Google Scholar 

  33. 33.

    Sullivan MP, Nieuwoudt MK, Bowmaker GA, Lam NYS, Truong D, Goldstone DC, Hartinger CG (2018) Unexpected arene ligand exchange results in the oxidation of an organoruthenium anticancer agent: the first X-ray structure of a protein-Ru(carbene) adduct. Chem Commun 54(48):6120–6123

    CAS  Article  Google Scholar 

  34. 34.

    McNae IW, Fishburne K, Habtemariam A, Hunter TM, Melchart M, Wang F, Walkinshaw MD, Sadler PJ (2004) Half-sandwich arene ruthenium(II)-enzyme complex. Chem Commun 10(16):1786–1787

    Article  Google Scholar 

  35. 35.

    Messori L, Merlino A (2014) Ruthenium metalation of proteins: the X-ray structure of the complex formed between NAMI-A and hen egg white lysozyme. Dalton Trans 43(16):6128–6131

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Sullivan MP, Nieuwoudt MK, Bowmaker GA, Lam NYS, Truong D, Goldstone DC, Hartinger CG (2018) Unexpected arene ligand exchange results in the oxidation of an organoruthenium anticancer agent: the first X-ray structure of a protein-Ru(carbene) adduct. Chem Commun 54(48):6120–6123 (Correction: Chem Commun (2019) 55(5):716)

    CAS  Article  Google Scholar 

  37. 37.

    Bajorath J, Hinrichs W, Saenger W (1988) The enzymatic activity of proteinase K is controlled by calcium. Eur J Biochem 176(2):441–447

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Russo Krauss I, Ferraro G, Pica A, Márquez JA, Helliwell JR, Merlino A (2017) Principles and methods used to grow and optimize crystals of protein-metallodrug adducts, to determine metal binding sites and to assign metal ligands. Metallomics 9(11):1534–1547

    CAS  PubMed  Article  Google Scholar 

  39. 39.

    Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, 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(4):235–242

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Kabsch W (2010) XDS. Acta Crystallogr Sect D Biol Crystallogr 66(2):125–132

    CAS  Article  Google Scholar 

  41. 41.

    McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40(4):658–674

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. 42.

    Vaney MC, Maignan S, Riès-Kautt M, Ducruix A (1996) High-resolution structure (1.33 Å) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from spaceHab-01 mission. Acta Crystallogr Sect D Biol Crystallogr 52(3):505–517

    CAS  Article  Google Scholar 

  43. 43.

    Wang J, Dauter M, Dauter Z (2006) What can be done with a good crystal and an accurate beamline? Acta Crystallogr D Biol Crystallogr 62(12):1475–1483

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    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 Crystallogr Sect D Biol Crystallogr 68(4):352–367

    CAS  Article  Google Scholar 

  45. 45.

    Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta Crystallogr Sect D Biol Crystallogr 60(12 I):2126–2132

    Article  CAS  Google Scholar 

  46. 46.

    Chen VB, Arendall Iii WB, 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(1):12–21

    CAS  PubMed  Article  Google Scholar 

  47. 47.

    Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Electrostatics of nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci 98(18):10037–10041

    CAS  PubMed  Article  Google Scholar 

  48. 48.

    Wang J, Dauter M, Alkire R, Joachimiak A, Dauter Z (2007) Triclinic lysozyme at 0.65 Å resolution. Acta Crystallogr Sect D Biol Crystallogr 63(12):1254–1268

    CAS  Article  Google Scholar 

  49. 49.

    Vocadlo DJ, Davies GJ, Laine R, Withers SG (2001) Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature 412(6849):835–838

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Betzel C, Gourinath S, Kumar P, Kaur P, Perbandt M, Eschenburg S, Singh TP (2001) Structure of a serine protease proteinase K from Tritirachium album limber at 0.98 Å resolution. Biochemistry 40(10):3080–3088

    CAS  PubMed  Article  Google Scholar 

  51. 51.

    Masuda T, Suzuki M, Inoue S, Song C, Nakane T, Nango E, Tanaka R, Tono K, Joti Y, Kameshima T, Hatsui T, Yabashi M, Mikami B, Nureki O, Numata K, Iwata S, Sugahara M (2017) Atomic resolution structure of serine protease proteinase K at ambient temperature. Sci Rep 7:45604

    PubMed  PubMed Central  Article  Google Scholar 

  52. 52.

    Zheng H, Cooper DR, Porebski PJ, Shabalin IG, Handing KB, Minor W (2017) CheckMyMetal: a macromolecular metal-binding validation tool. Acta Crystallogr Sect D Struct Biol 73:223–233

    CAS  Article  Google Scholar 

  53. 53.

    Bajorath J, Raghunathan S, Hinrichs W, Saenger W (1989) Long-range structural changes in proteinase K triggered by calcium ion removal. Nature 337(6206):481–484

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Muller A, Hinrichs W, Wolf WM, Saenger W (1994) Crystal structure of calcium-free proteinase K at 1.5-Å resolution. J Biol Chem 269(37):23108–23111

    CAS  PubMed  Google Scholar 

  55. 55.

    Santos-Silva T, Mukhopadhyay A, Seixas JD, Bernardes GJL, Romão CC, Romão MJ (2011) CORM-3 reactivity toward proteins: the crystal structure of a Ru(II) dicarbonyl-lysozyme complex. J Am Chem Soc 133(5):1192–1195

    CAS  PubMed  Article  Google Scholar 

  56. 56.

    Seixas JD, Santos MFA, Mukhopadhyay A, Coelho AC, Reis PM, Veiros LF, Marques AR, Penacho N, Gonçalves AML, Romão MJ, Bernardes GJL, Santos-Silva T, Romão CC (2015) A contribution to the rational design of Ru(CO)3Cl2L complexes for in vivo delivery of CO. Dalton Trans 44(11):5058–5075

    CAS  PubMed  Article  Google Scholar 

  57. 57.

    Tamasi G, Merlino A, Scaletti F, Heffeter P, Legin AA, Jakupec MA, Berger W, Messori L, Keppler BK, Cini R (2017) {Ru(CO)x}-Core complexes with benzimidazole ligands: synthesis, X-ray structure and evaluation of anticancer activity in vivo. Dalton Trans 46(9):3025–3040

    CAS  PubMed  Article  Google Scholar 

  58. 58.

    Pontillo N, Ferraro G, Messori L, Tamasi G, Merlino A (2017) Ru-Based CO releasing molecules with azole ligands: interaction with proteins and the CO release mechanism disclosed by X-ray crystallography. Dalton Trans 46(29):9621–9629

    CAS  PubMed  Article  Google Scholar 

  59. 59.

    Messori L, Marzo T, Sanches RNF, Hanif Ur R, De Oliveira SD, Merlino A (2014) Unusual structural features in the lysozyme derivative of the tetrakis(acetato)chloridodiruthenium(ii, iii) complex. Angew Chem Int Ed 53(24):6172–6175

    CAS  Article  Google Scholar 

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Acknowledgements

We would like to thank Prof. Enzo Alessio and the reviewers for their constructive comments on the manuscript. We would also like to thank the beamline scientists at the synchrotron facilities of the Swiss Light Source at PSI, (Villigen, Switzerland) and of PETRA III at EMBL (Hamburg, Germany) for their assistance during X-ray data collection. This work was supported by the project “INSPIRED” (Grant MIS 5002550).

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Correspondence to Petros Giastas or Athanasios Papakyriakou.

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Data deposition: Atomic coordinates and structure factors have been deposited in the Protein Data Bank, https://www.rcsb.org/ (PDB ID codes 6TVL and 6TXG).

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Chiniadis, L., Bratsos, I., Bethanis, K. et al. High-resolution crystal structures of a “half sandwich”-type Ru(II) coordination compound bound to hen egg-white lysozyme and proteinase K. J Biol Inorg Chem 25, 635–645 (2020). https://doi.org/10.1007/s00775-020-01786-z

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Keywords

  • Ruthenium anticancer compounds
  • X-ray crystallography
  • Lysozyme (HEWL)
  • Proteinase K
  • Protein ruthenation