Bridging solvent molecules mediate RNase A – Ligand binding

Autoři: Stefan M. Ivanov aff001;  Ivan Dimitrov aff001;  Irini A. Doytchinova aff001
Působiště autorů: Faculty of Pharmacy, Medical University of Sofia, Sofia, Bulgaria aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224271


Due to its high catalytic activity and readily available supply, ribonuclease A (RNase A) has become a pivotal enzyme in the history of protein science. Moreover, this great interest has carried over to computational chemistry and molecular dynamics, where RNase A has become a model system for various types of studies, all the while being an important drug design target in its own right. Here, we present a detailed molecular dynamics study of RNase–ligand binding involving 22 compounds, spanning nearly five orders of magnitude in affinity, and totaling 8.8 μs of sampling with the standard Amber parameters and an additional 8.8 μs of sampling with a modified potential. We show that short-lived, solvent-mediated bridging interactions are crucial to RNase–ligand binding. We characterize the behavior of bridging solvent molecules, uncovering a power-law dependence between the lifetime of a solvent bridge and the probability of its occurrence. We also demonstrate that from an energetic perspective, bridging solvent in RNase A–ligand binding behaves like part of the enzyme, rather than the ligands. Moreover, we describe an automated pipeline for the detection and processing of bridging interactions, and offer an independent assessment of the performance of the state-of-the-art fixed-charge force fields. Thus, our work has broad implications for drug design and computational chemistry in general.

Klíčová slova:

Crystal structure – Lysine – Oxygen – Phosphates – Protein interactions – Ribonucleases – Sodium – Arginine


1. Rosenberg HF. RNase A ribonucleases and host defense: an evolving story. J Leukoc Biol. 2008;83(5):1079–87. doi: 10.1189/jlb.1107725 18211964

2. Köten B, Simanski M, Gläser R, Podschun R, Schröder JM, Harder J. RNase 7 contributes to the cutaneous defense against Enterococcus faecium. PLoS One. 2009;4(7).

3. Dyer KD, Rosenberg HF. The RNase a superfamily: Generation of diversity and innate host defense. Mol Divers. 2006;10(4):585–97. doi: 10.1007/s11030-006-9028-2 16969722

4. Cuchillo CM, Nogués MV, Raines RT. Bovine pancreatic ribonuclease: Fifty years of the first enzymatic reaction mechanism. Biochemistry. 2011;50(37):7835–41. doi: 10.1021/bi201075b 21838247

5. Marshall GR, Feng JA, Kuster DJ. Back to the future: Ribonuclease A. Biopolym—Pept Sci Sect. 2008;90(3):259–77.

6. Jenkins CL, Thiyagarajan N, Sweeney RY, Guy MP, Kelemen BR, Acharya KR, et al. Binding of non-natural 3’-nucleotides to ribonuclease A. FEBS J. 2005;272(3):744–55. doi: 10.1111/j.1742-4658.2004.04511.x 15670155

7. Leonidas DD, Shapiro R, Irons LI, Russo N, Acharya KR. Toward rational design of ribonuclease inhibitors: High-resolution crystal structure of a ribonuclease a complex with a potent 3’,5’- pyrophosphate-linked dinucleotide inhibitor. Biochemistry. 1999;38(32):10287–97. doi: 10.1021/bi990900w 10441122

8. Zegers I, Maes D, Poortmans F, Palmer R, Wyns L, Zegers I, et al. The structures of RNase A complexed with 3 ‘-CMP and d(CpA): Active site conformation and conserved water molecules. Protein Sci. 1994;3(12):2322–39. doi: 10.1002/pro.5560031217 7756988

9. Barillari C, Taylor J, Viner R, Essex JW. Classification of Water Molecules in Protein Binding Sites. J Am Chem Soc. 2007;129(9):2577–87. doi: 10.1021/ja066980q 17288418

10. Sahai MA, Biggin PC. Quantifying water-mediated protein-ligand interactions in a glutamate receptor: A DFT study. J Phys Chem B. 2011;115(21):7085–96. doi: 10.1021/jp200776t 21545106

11. Leonidas DD, Chavali GB, Oikonomakos NG, Chrysina ED, Kosmopoulou MN, Vlassi M, et al. High-resolution crystal structures of ribonuclease A complexed with adenylic and uridylic nucleotide inhibitors. Implications for structure-based design of ribonucleolytic inhibitors. Protein Sci. 2003;12(11):2559–74. doi: 10.1110/ps.03196603 14573867

12. Jardine AM, Leonidas DD, Jenkins JL, Park C, Raines RT, Acharya KR, et al. Cleavage of 3′,5′-Pyrophosphate-Linked Dinucleotides by Ribonuclease A and Angiogenin. Biochemistry. 2001;40(34):10262–72. doi: 10.1021/bi010888j 11513604

13. Leonidas DD, Shapiro R, Irons LI, Russo N, Acharya KR. Crystal structures of ribonuclease a complexes with 5’- diphosphoadenosine 3’-phosphate and 5’-diphosphoadenosine 2’-phosphate at 1.7 Å resolution. Biochemistry. 1997;36(18):5578–88. doi: 10.1021/bi9700330 9154942

14. Parmenopoulou V, Chatzileontiadou DSM, Manta S, Bougiatioti S, Maragozidis P, Gkaragkouni D, et al. Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis, biochemical, and structural evaluation. Bioorg Med Chem. 2012;20(24):7184–93. doi: 10.1016/j.bmc.2012.09.067 23122937

15. Samanta A, Leonidas DD, Dasgupta S, Pathak T, Zographos SE. Morpholino, Piperidino, and Pyrrolidino Derivatives of Pyrimidine Nucleosides as Inhibitors of Ribonuclease A: Synthesis, Biochemical, and Crystallographic Evaluation. J Med Chem. 2009;54(2):932–42.

16. Wang JM, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and Testing of a General Amber Force Field. J Comput Chem. 2004;25(9):1157–74. doi: 10.1002/jcc.20035 15116359

17. Sliwoski G, Kothiwale S, Meiler J, Lowe EW. Computational Methods in Drug Discovery. PharmacolRev. 2014;66(1):334–95.

18. Wang R, Fang X, Lu Y, Wang S. The PDBbind Database: Collection of Binding Affinities for Protein−Ligand Complexes with Known Three-Dimensional Structures. J Med Chem. 2004;47(12):2977–80. doi: 10.1021/jm030580l 15163179

19. Wang R, Fang X, Lu Y, Yang CY, Wang S. The PDBbind database: Methodologies and updates. J Med Chem. 2005;48(12):4111–9. doi: 10.1021/jm048957q 15943484

20. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. J Chem Phys. 1983;79(1983):926.

21. Joung IS, Cheatham TE. Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations. J Phys Chem B. 2008;112(30):9020–41. doi: 10.1021/jp8001614 18593145

22. Jakalian A, Bush BL, Jack DB, Bayly CI. Fast, efficient generation of high-quality atomic charges. AM1-BCC model: I. Method. J Comput Chem. 2000;21(2):132–46.

23. Berendsen HJ. C, Postma JPM, van Gunsteren WF, DiNola A, Haak JR. Molecular dynamics with coupling to an external bath. J Chem Phys. 1984;81(1984):3684–90.

24. Adelman SA, Doll JD. Generalized Langevin equation approach for atom/solid‐surface scattering: Collinear atom/harmonic chain model. J Chem Phys. 1974;61(10):4242–5.

25. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J Chem Theory Comput. 2015;11(8):3696–3713. doi: 10.1021/acs.jctc.5b00255 26574453

26. Darden T, York D, Pedersen L. Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems. J Chem Phys. 1993;98(1993):10089.

27. Ciccotti G, Ryckaert JP. Molecular dynamics simulation of rigid molecules. Comput Phys Reports. 1986;4(6):346–92.

28. Roe DR, Cheatham TE. PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J Chem Theory Comput. 2013;9(7):3084–95. doi: 10.1021/ct400341p 26583988

29. Miller BR, Mcgee TD, Swails JM, Homeyer N, Gohlke H, Roitberg AE. An Efficient Program for End-State Free Energy Calculations. J Chem Theory Comput. 2012;8(9):pp 3314–3321. doi: 10.1021/ct300418h 26605738

30. Homeyer N, Gohlke H. Free Energy Calculations by the Molecular Mechanics Poisson−Boltzmann Surface Area Method. Mol Inform. 2012;31:114–22. doi: 10.1002/minf.201100135 27476956

31. Gohlke H, Case DA. Converging Free Energy Estimates: MM-PB(GB)SA Studies on the Protein–Protein Complex Ras–Raf. J Comput Chem. 2003;25(2):238–50.

32. Brandsdal BO, Österberg F, Almlöf M, Freierberg I, Luzhkov VB, Åqvist J. Free Energy Calculations and Ligand Binding. Adv Protein Chem. 2003;66:123–58. 14631818

33. Warwicker J, Watson HC. Calculation of the electric potential in the active site cleft due to α-helix dipoles. J Mol Biol. 1982;157(4):671–9. doi: 10.1016/0022-2836(82)90505-8 6288964

34. Tan C, Tan YH, Luo R. Implicit nonpolar solvent models. J Phys Chem B. 2007;111(2):12263–74.

35. Pratt L, Chandler D. Effective intramolecular potentials for molecular bromine in argon. Comparison of theory with simulation. J Chem Phys. 1980;4045(1980).

36. Huang DM, Chandler D. The hydrophobic effect and the influence of solute-solvent attractions. J Phys Chem B. 2002;106:2047–53.

37. Koehl P. Electrostatics calculations: latest methodological advances. CurrOpin Struct Biol. 2006;16(2):142–51.

38. Onufriev A, Bashford D, Case D a. Exploring Protein Native States and Large-Scale Conformational Changes with a Modified Generalized Born Model. Proteins Struct Funct Genet. 2004;55(2):383–94. doi: 10.1002/prot.20033 15048829

39. Weiser Jörg, Peter S. Shenkin WCS. Approximate Atomic Surfaces from Linear Combinations of Pairwise. 1999;20(2):217–30.

40. Meirovitch H, Cheluvaraja S, White RP. Methods for calculating the entropy and free energy and their application to problems involving protein flexibility and ligand binding. Curr Protein Pept Sci. 2009;10(3):229–43. 19519453

41. Huber RG, Fuchs JE, vonGrafenstein S, Laner M, Wallnoefer HG, Abdelkader N, et al. Entropy from state probabilities: hydration entropy of cations. J Phys Chem B. 2013;117(1):6466–72.

42. Horovitz A, Serrano L, Avron B, Bycroft M, Fersht AR. Strength and co-operativity of contributions to surface salt bridges to protein stability. J Mol Biol. 1990;216(4):1031–1044. doi: 10.1016/S0022-2836(99)80018-7 2266554

43. Horovitz A, Fersht A. R. Co-operative Interactions during Protein Folding. J Mol Biol. 1992;224(3):733–40. doi: 10.1016/0022-2836(92)90557-z 1569552

44. Ivanov SM, Huber RG, Warwicker J, Bond PJ. Energetics and Dynamics Across the Bcl-2-Regulated Apoptotic Pathway Reveal Distinct Evolutionary Determinants of Specificity and Affinity. Structure. 2016;24(11):2024–33. doi: 10.1016/j.str.2016.09.006 27773689

45. Ivanov SM, Huber RG, Alibay I, Warwicker J, Bond PJ. Energetic Fingerprinting of Ligand Binding to Paralogous Proteins: The Case of the Apoptotic Pathway. J Chem Inf Model. 2019;59(1):245–61. doi: 10.1021/acs.jcim.8b00765 30582811

46. Day CL, Smits C, Fan FC, Lee EF, Fairlie WD, Hinds MG. Structure of the BH3 Domains from the p53-Inducible BH3-Only Proteins Noxa and Puma in Complex with Mcl-1. J Mol Biol. 2008;380(5):958–71. doi: 10.1016/j.jmb.2008.05.071 18589438

47. Maffucci I, Contini A. Explicit ligand hydration shells improve the correlation between MM-PB/GBSA binding energies and experimental activities. J Chem Theory Comput. 2013;9(6):2706–17. doi: 10.1021/ct400045d 26583864

48. Tame JRH, Sleigh SH, Wilkinson AJ, Ladbury JE. The role of water in sequence-independent ligand binding by an oligopeptide transporter protein. Nat Struct Biol. 1996;3(12):998–1001. 8946852

49. Ladbury JE. Just add water! The effect of water on the specificity of protein-ligand binding sites and its potential application to drug design. Chem Biol. 1996;3(12):973–80. 9000013

50. Beach H, Cole R, Gill ML, Loria JP. Conservation of μs-ms enzyme motions in the apo- and substrate-mimicked state. J Am Chem Soc. 2005;127(25):9167–76. doi: 10.1021/ja0514949 15969595

51. Zheng H, Cooper DR, Porebski PJ, Shabalin IG, Handing KB, Minor W. CheckMyMetal: A macromolecular metal-binding validation tool. Acta Crystallogr Sect D Struct Biol. 2017;73:223–33.

52. Tereshko V. Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments. Nucleic Acids Res. 2001;29(5):1208–15. doi: 10.1093/nar/29.5.1208 11222771

53. Makarov VA, Andrews BK, Smith PE, Pettitt BM. Residence times of water molecules in the hydration sites of myoglobin. Biophys J. 2000;79(6):2966–74. doi: 10.1016/S0006-3495(00)76533-7 11106604

54. Henchman RH, McCammon JA. Extracting hydration sites around proteins from explicit water simulations. J Comput Chem. 2002;23(9):861–9. doi: 10.1002/jcc.10074 11984847

55. Halle B, Helliwell JR, Kornyshev A, Engberts JBFN. Protein hydration dynamics in solution: A critical survey. Philos Trans R Soc B Biol Sci. 2004;359(1448):1207–24.

56. Persson F, Söderhjelm P, Halle B. How proteins modify water dynamics. J Chem Phys. 2018;148(21).

57. Ivanov SM, Cawley A, Huber RG, Bond PJ, Warwicker J. Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold. PLoS One. 2017;12(10):1–16.

58. Knapp B, Ospina L, Deane CM. Avoiding False Positive Conclusions in Molecular Simulation: The Importance of Replicas. J Chem Theory Comput. 2018;14(12):6127–38. doi: 10.1021/acs.jctc.8b00391 30354113

59. Yoo J, Aksimentiev A. Improved Parameterization of Amine-Carboxylate and Amine-Phosphate Interactions for Molecular Dynamics Simulations Using the CHARMM and AMBER Force Fields. J Chem Theory Comput. 2016;12(1):430–43. doi: 10.1021/acs.jctc.5b00967 26632962

60. Yoo J, Aksimentiev A. New tricks for old dogs: improving the accuracy of biomolecular force fields by pair-specific corrections to non-bonded interactions. Phys Chem Chem Phys. 2018;20:8432–49. doi: 10.1039/C7CP08185E 29547221

61. Nerenberg PS, Jo B, So C, Tripathy A, Head-Gordon T. Optimizing Solute-Water van der Waals Interactions To Reproduce Solvation Free Energies. J Phys Chem B. 2012;116(15):4524–34. doi: 10.1021/jp2118373 22443635

62. Piana S, Klepeis JL, Shaw DE. Assessing the accuracy of physical models used in protein-folding simulations: Quantitative evidence from long molecular dynamics simulations. CurrOpin Struct Biol. 2014;24(1):98–105.

63. Yoo J, Aksimentiev A. Improved Parametrization of Li+, Na+, K+, and Mg2+ ions for All-Atom Molecular Dynamics Simulations of Nucleic Acid Systems. J Phys Chem Lett. 2012;3(1):45–50.

64. Vassetti D, Pagliai M, Procacci P. Assessment of GAFF2 and OPLS-AA General Force Fields in Combination with the Water Models TIP3P, SPCE, and OPC3 for the Solvation Free Energy of Druglike Organic Molecules. J Chem Theory Comput. 2019;15(3):1983–95.65.

65. Schuber F. Influence of polyamines on membrane functions. Biochem J. 1989;260(1):1–10. doi: 10.1042/bj2600001 2673211

66. Lightfoot HL, Hall J. Endogenous polyamine function—The RNA perspective. Nucleic Acids Res. 2014;42(18):11275–90.</References> doi: 10.1093/nar/gku837 25232095

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