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Journal of Computer-Aided Molecular Design

, Volume 32, Issue 12, pp 1375–1388 | Cite as

Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase: insights from molecular dynamics simulations

  • Stefan A. P. Lenz
  • Stacey D. Wetmore
Article
  • 28 Downloads

Abstract

Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine–uridine nucleoside hydrolase (CU–NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU–NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU–NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU–NH are identified. Regardless of the wild-type or mutant CU–NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU–NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU–NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.

Graphical Abstract

Keywords

Molecular dynamics Cytidine-uridine nucleoside hydrolase Inosine-uridine nucleoside hydrolase Substrate binding Hydrolysis of inosine 

Abbreviations

A

Adenosine

AAG

Alkyladenine DNA glycosylase

C

Cytidine

CU–NH

Cytidine–uridine nucleoside hydrolase

G

Guanosine

I

Inosine

IAG–NH

Inosine–adenosine–guanosine nucleoside hydrolase

IG–NH

Inosine–guanosine nucleoside hydrolase

IU–NH

Inosine–uridine nucleoside hydrolase

MCPB

Metal center parameter builder

MD

Molecular dynamics

NH

Nucleoside hydrolase

pAPIR

p-Aminophenyliminoribitol

PDB

Protein data bank

RESP

Restrained electrostatic potential

rms

Root-mean-square

rmsd

Root-mean-square deviation

U

Uridine

UDG

Uracil DNA glycosylase

X

Xanthosine

Notes

Acknowledgements

Computational resources from the New Upscale Cluster for Lethbridge to Enable Innovative Chemistry (NUCLEIC) and those provided by Westgrid and Compute/Calcul Canada are greatly appreciated.

Funding

Support for this research was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC, Grant No. 2016–04568), the Canada Foundation for Innovation (Grant No. 22770) and the Board of Governors Research Chair Program at the University of Lethbridge. S.A.P.L. acknowledges NSERC (CGS-D), Alberta Innovates-Technology Futures (AI-TF) and the University of Lethbridge for student scholarships.

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interests.

Supplementary material

10822_2018_178_MOESM1_ESM.docx (19.4 mb)
Supplementary material 1 (DOCX 19888 KB)

References

  1. 1.
    Barrett MP, Croft SL (2012) Management of trypanosomiasis and leishmaniasis. Br Med Bull 104:175–196CrossRefGoogle Scholar
  2. 2.
    el Kouni MH (2003) Potential chemotherapeutic targets in the purine metabolism of parasites. Pharmacol Ther 99:283–309CrossRefGoogle Scholar
  3. 3.
    Delespaux V, de Koning HP (2007) Drugs and drug resistance in African trypanosomiasis. Drug Resist Updat 10:30–50CrossRefGoogle Scholar
  4. 4.
    Kennedy PGE (2013) Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol 12:186–194CrossRefGoogle Scholar
  5. 5.
    Minodier P, Parola P (2007) Cutaneous leishmaniasis treatment, Travel Med. Infect Dis 5:150–158Google Scholar
  6. 6.
    Garcia MN, O’Day S, Fisher-Hoch S, Gorchakov R, Patino R, Arroyo F, Laing TP, Lopez ST, Ingber JE, Jones A, K. M., et al (2016) One health interactions of Chagas disease vectors, canid hosts, and human residents along the Texas-Mexico border. PLoS Negl Trop Dis 10:e0005074CrossRefGoogle Scholar
  7. 7.
    Garcia MN, Woc-Colburn L, Aguilar D, Hotez PJ, Murray KO (2015) Historical perspectives on the epidemiology of human Chagas disease in Texas and recommendations for enhanced understanding of clinical Chagas disease in the southern United States. PLoS Negl Trop Dis 9:e0003981CrossRefGoogle Scholar
  8. 8.
    Demers E, Forrest DM, Weichert GE (2013) Cutaneous leishmaniasis in a returning traveller. Can Med Assoc J 185:681–683CrossRefGoogle Scholar
  9. 9.
    Boitz JM, Ullman B, Jardim A, Carter NS (2012) Purine salvage in Leishmania: complex or simple by design? Trends Parasitol 28:345–352CrossRefGoogle Scholar
  10. 10.
    Wilson ZN, Gilroy CA, Boitz JM, Ullman B, Yates PA (2012) Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani. J Biol Chem 287:12759–12770CrossRefGoogle Scholar
  11. 11.
    Carter NS, Yates P, Arendt CS, Boitz JM, Ullman B (2008) Purine and pyrimidine metabolism in Leishmania. In: Majumder H (ed) Drug targets in kinetoplastid parasites. Springer, New York, pp. 141–154.CrossRefGoogle Scholar
  12. 12.
    Hammond DJ, Gutteridge WE (1984) Purine and pyrimidine metabolism in the Trypanosomatidae. Mol Biochem Parasitol 13:243–261CrossRefGoogle Scholar
  13. 13.
    Murkin AS, Moynihan MM (2014) Transition-state-guided drug design for treatment of parasitic neglected tropical diseases. Curr Med Chem 21:1781–1793CrossRefGoogle Scholar
  14. 14.
    Degano M, Gopaul DN, Scapin G, Schramm VL, Sacchettini JC (1996) Three-dimensional structure of the inosine-uridine nucleoside N-ribohydrolase from Crithidia fasciculata. Biochemistry 35:5971–5981CrossRefGoogle Scholar
  15. 15.
    Horenstein BA, Zabinski RF, Schramm VL (1993) A new class of C-nucleoside analogues. 1-(S)-aryl-1,4-dideoxy-1,4-imino-d-ribitols, transition state analogue inhibitors of nucleoside hydrolase. Tetrahedron Lett 34:7213–7216CrossRefGoogle Scholar
  16. 16.
    Shi WX, Schramm VL, Almo SC (1999) Nucleoside hydrolase from Leishmania major—cloning, expression, catalytic properties, transition state inhibitors, and the 2.5-Å crystal structure. J Biol Chem 274:21114–21120CrossRefGoogle Scholar
  17. 17.
    Evans GB, Furneaux RH, Gainsford GJ, Schramm VL, Tyler PC (2000) Synthesis of transition state analogue inhibitors for purine nucleoside phosphorylase and N-riboside hydrolases. Tetrahedron 56:3053–3062CrossRefGoogle Scholar
  18. 18.
    Berg M, Van der Veken P, Goeminne A, Haemers A, Augustyns K (2010) Inhibitors of the purine salvage pathway: a valuable approach for antiprotozoal chemotherapy? Curr Med Chem 17:2456–2481CrossRefGoogle Scholar
  19. 19.
    Berg M, Kohl L, Van der Veken P, Joossens J, Al-Salabi MI, Castagna V, Giannese F, Cos P, Versees W, Steyaert J et al (2010) Evaluation of nucleoside hydrolase inhibitors for treatment of African trypanosomiasis. Antimicrob Agents Chemother 54:1900–1908CrossRefGoogle Scholar
  20. 20.
    Versees W, Goeminne A, Berg M, Vandemeulebroucke A, Haemers A, Augustyns K, Steyaert J (2009) Crystal structures of T. vivax nucleoside hydrolase in complex with new potent and specific inhibitors. Biochim Biophys Acta Proteins Proteom 1794:953–960CrossRefGoogle Scholar
  21. 21.
    Goeminne A, Berg M, McNaughton M, Bal G, Surpateanu G, Van der Veken P, De Prol S, Versees W, Steyaert J, Haemers A et al (2008) N-arylmethyl substituted iminoribitol derivatives as inhibitors of a purine specific nucleoside hydrolase. Biorg Med Chem 16:6752–6763CrossRefGoogle Scholar
  22. 22.
    Goeminne A, McNaughton M, Bal G, Surpateanu G, Van der Veken P, De Prol S, Versees W, Steyaert J, Haemers A, Augustyns K (2008) Synthesis and biochemical evaluation of guanidino-alkyl-ribitol derivatives as nucleoside hydrolase inhibitors. Eur J Med Chem 43:315–326CrossRefGoogle Scholar
  23. 23.
    Renno MN, Franca C, Nico D, Palatnik-de-Sousa CB, Tinoco LW, Figueroa-Villar JD (2012) Kinetics and docking studies of two potential new inhibitors of the nucleoside hydrolase from Leishmania donovani. Eur J Med Chem 56:301–307CrossRefGoogle Scholar
  24. 24.
    Franca TCC, Rocha MdRM, Reboredo BM, Renno MN, Tinoco LW, Figueroa-Villar JD (2008) Design of inhibitors for nucleoside hydrolase from Leishmania donovani using molecular dynamics studies. J Braz Chem Soc 19:64–73CrossRefGoogle Scholar
  25. 25.
    Goeminne A, McNaughton M, Bal G, Surpateartu G, Van der Veken P, De Prol S, Versees W, Steyaert J, Apers S, Haemers A et al (2007) 1,2,3-triazolylalkylribitol derivatives as nucleoside hydrolase inhibitors. Bioorg Med Chem Lett 17:2523–2526CrossRefGoogle Scholar
  26. 26.
    Versees W, Steyaert J (2003) Catalysis by nucleoside hydrolases. Curr Opin Struct Biol 13:731–738CrossRefGoogle Scholar
  27. 27.
    Vandemeulebroucke A, De Vos S, Van Holsbeke E, Steyaert J, Versees W (2008) A flexible loop as a functional element in the catalytic mechanism of nucleoside hydrolase from Trypanosoma vivax. J Biol Chem 283:22272–22282CrossRefGoogle Scholar
  28. 28.
    Versees W, Decanniere K, Van Holsbeke E, Devroede N, Steyaert J (2002) Enzyme-substrate interactions in the purine-specific nucleoside hydrolase from Trypanosoma vivax. J Biol Chem 277:15938–15946CrossRefGoogle Scholar
  29. 29.
    Versees W, Decanniere K, Pelle R, Depoorter J, Brosens E, Parkin DW, Steyaert J (2001) Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax. J Mol Biol 307:1363–1379CrossRefGoogle Scholar
  30. 30.
    Arivett B, Farone M, Masiragani R, Burden A, Judge S, Osinloye A, Minici C, Degano M, Robinson M, Kline P (2014) Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli. Biochim Biophys Acta Proteins Proteom 1844:656–662CrossRefGoogle Scholar
  31. 31.
    Giannese F, Berg M, Van der Veken P, Castagna V, Tornaghi P, Augustyns K, Degano M (2013) Structures of purine nucleosidase from Trypanosoma brucei bound to isozyme-specific trypanocidals and a novel metalorganic inhibitor. Acta Crystallogr Sect D 69:1553–1566CrossRefGoogle Scholar
  32. 32.
    Iovane E, Giabba B, Muzzolini L, Matafora V, Fornili A, Minici C, Giannese F, Degano M (2008) Structural basis for substrate specificity in group I nucleoside hydrolases. Biochemistry 47:4418–4426CrossRefGoogle Scholar
  33. 33.
    Giabbai B, Degano M (2004) Crystal structure to 1.7 angstrom of the Escherichia coli pyrimidine nucleoside hydrolase YeiK, a novel candidate for cancer gene therapy. Structure 12:739–749CrossRefGoogle Scholar
  34. 34.
    Gopaul DN, Meyer SL, Degano M, Sacchettini JC, Schramm VL (1996) Inosine-uridine nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue. Biochemistry 35:5963–5970CrossRefGoogle Scholar
  35. 35.
    Vandemeulebroucke A, Minici C, Bruno I, Muzzolini L, Tornaghi P, Parkin DW, Versees W, Steyaert J, Degano M (2010) Structure and mechanism of the 6-oxopurine nucleosidase from Trypanosoma brucei brucei. Biochemistry 49:8999–9010CrossRefGoogle Scholar
  36. 36.
    Degano M, Almo SC, Sacchettini JC, Schramm VL (1998) Trypanosomal nucleoside hydrolase. A novel mechanism from the structure with a transition-state inhibitor. Biochemistry 37:6277–6285CrossRefGoogle Scholar
  37. 37.
    Horenstein BA, Parkin DW, Estupinan B, Schramm VL (1991) Transition-state analysis of nucleoside hydrolase from Crithidia fasciculata. Biochemistry 30:10788–10795CrossRefGoogle Scholar
  38. 38.
    Parkin DW, Schramm VL (1995) Binding modes for substrate and a proposed transition-state analog of protozoan nucleoside hydrolase. Biochemistry 34:13961–13966CrossRefGoogle Scholar
  39. 39.
    Mazumder D, Bruice TC (2002) Exploring nucleoside hydrolase catalysis in silico: molecular dynamics study of enzyme-bound substrate and transition state. J Am Chem Soc 124:14591–14600CrossRefGoogle Scholar
  40. 40.
    Fornili A, Giabbai B, Garau G, Degano M (2010) Energy landscapes associated with macromolecular conformational changes from endpoint structures. J Am Chem Soc 132:17570–17577CrossRefGoogle Scholar
  41. 41.
    Fan F, Chen N, Wang Y, Wu R, Cao Z (2018) QM/MM and MM MD simulations on the pyrimidine-specific nucleoside hydrolase: a comprehensive understanding of enzymatic hydrolysis of uridine. J Phys Chem B 122:1121–1131CrossRefGoogle Scholar
  42. 42.
    Versees W, Loverix S, Vandemeulebroucke A, Geerlings P, Steyaert J (2004) Leaving group activation by aromatic stacking: an alternative to general acid catalysis. J Mol Biol 338:1–6CrossRefGoogle Scholar
  43. 43.
    Mazumder-Shivakumar D, Bruice TC (2005) Computational study of IAG-nucleoside hydrolase: determination of the preferred ground state conformation and the role of active site residues. Biochemistry 44:7805–7817CrossRefGoogle Scholar
  44. 44.
    Wu R, Gong W, Ting L, Zhang Y, Cao Z (2012) QM/MM molecular dynamics study of purine-specific nucleoside hydrolase. J Phys Chem B 116:1984–1991CrossRefGoogle Scholar
  45. 45.
    Guimaraes AP, Oliveira AA, da Cunha EFF, Ramalho TC, Franco TCC (2011) Analysis of Bacillus anthracis nucleoside hydrolase via in silico docking with inhibitors and molecular dynamics simulation. J Mol Model 17:2939–2951CrossRefGoogle Scholar
  46. 46.
    Mancini DT, Matos KS, da Cunha EFF, Assis TM, Guimaraes AP, Franca TCC, Ramalho TC (2012) Molecular modeling studies on nucleoside hydrolase from the biological warfare agent Brucella suis. J Biomol Struct Dyn 30:125–136CrossRefGoogle Scholar
  47. 47.
    Anandakrishnan R, Aguilar B, Onufriev AV (2012) H++ 3.0: Automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res 40:W537–W541CrossRefGoogle Scholar
  48. 48.
    Case DA, Darden TA, Cheatham TE, Simmerling CL, Wang J, Duke J, Luo R, Crowley M, Walker RC, Zhang W et al (2008) Amber Tools, Version 1.0 edn. University of California, San FranciscoGoogle Scholar
  49. 49.
    Wang JM, Wolf RM, Caldwell JW, Kollman PA, Case DA (2004) Development and testing of a general Amber force field. J Comput Chem 25:1157–1174CrossRefGoogle Scholar
  50. 50.
    Li P, Merz KM (2016) MCPB.Py: a python based metal center parameter builder. J Chem Inf Model 56:599–604CrossRefGoogle Scholar
  51. 51.
    Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA et al (2016) Gaussian 09, Revision D. 01. Gaussian, Inc., WallingfordGoogle Scholar
  52. 52.
    Seminario JM (1996) Calculation of intramolecular force fields from second-derivative tensors. Int J Quantum Chem 60:1271–1277CrossRefGoogle Scholar
  53. 53.
    Salomon-Ferrer R, Götz AW, Poole D, Le Grand S, Walker RC (2013) Routine microsecond molecular dynamics simulations with Amber on GPUs. 2. Explicit solvent particle mesh ewald. J Chem Theory Comput 9:3878–3888CrossRefGoogle Scholar
  54. 54.
    Le Grand S, Götz AW, Walker RC (2013) SPFP: Speed without compromise—a mixed precision model for GPU accelerated molecular dynamics simulations. Comput Phys Commun 184:374–380CrossRefGoogle Scholar
  55. 55.
    Roe DR, Cheatham TE (2013) PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J Chem Theory Comput 9:3084–3095CrossRefGoogle Scholar
  56. 56.
    Altona C, Sundaralingam M (1972) Conformational analysis of the sugar ring in nucleosides and nucleotides. New description using the concept of pseudorotation. J Am Chem Soc 94:8205–8212CrossRefGoogle Scholar
  57. 57.
    Lenz SAP, Kohout JD, Wetmore SD (2016) Hydrolytic glycosidic bond cleavage in RNA nucleosides: effects of the 2′-hydroxy group and acid–base catalysis. J Phys Chem B 120:12795–12806CrossRefGoogle Scholar
  58. 58.
    Wilson KA, Wells RA, Abendong MN, Anderson CB, Kung RW, Wetmore SD (2015) Landscape of π–π and sugar–π contacts in DNA–protein interactions. J Biomol Struct Dyn 34:184–200CrossRefGoogle Scholar
  59. 59.
    Wilson KA, Kellie JL, Wetmore SD (2014) DNA-protein pi-interactions in nature: abundance, structure, composition and strength of contacts between aromatic amino acids and DNA nucleobases or deoxyribose sugar. Nucleic Acids Res 42:6726–6741CrossRefGoogle Scholar
  60. 60.
    Lenz SAP, Wetmore SD (2016) Evaluating the substrate selectivity of alkyladenine DNA glycosylase: the synergistic interplay of active site flexibility and water reorganization. Biochemistry 55:798–808CrossRefGoogle Scholar
  61. 61.
    Rutledge LR, Wetmore SD (2011) Modeling the chemical step utilized by human alkyladenine DNA glycosylase: a concerted mechanism aids in selectively excising damaged purines. J Am Chem Soc 133:16258–16269CrossRefGoogle Scholar
  62. 62.
    Bashford D, Karplus M (1990) Pka’s of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry 29:10219–10225CrossRefGoogle Scholar
  63. 63.
    Parikh SS, Mol CD, Slupphaug G, Bharati S, Krokan HE, Tainer JA (1998) Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO J 17:5214–5226CrossRefGoogle Scholar

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© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of LethbridgeLethbridgeCanada

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