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Phosphorylation and ATP-binding induced conformational changes in the PrkC, Ser/Thr kinase from B. subtilis

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Abstract

Recent studies on the PrkC, serine-threonine kinase show that that the enzyme is located at the inner membrane of endospores and is responsible for triggering spore germination. The activity of the protein increases considerably after phosphorylation of four threonine residues placed on the activation loop and one serine placed in the C-terminal lobe of the PrkC. The molecular relationship between phosphorylation of these residues and enzyme activity is not known. In this work molecular dynamics simulation is performed on four forms of the protein kinase PrkC from B. subtilis—phosphorylated or unphosphorylated; with or without ATP bound—in order to gain insight into phosphorylation and ATP binding on the conformational changes and functions of the protein kinase. Our results show how phosphorylation, as well as the presence of ATP, is important for the activity of the enzyme through its molecular interaction with the catalytic core residues. Three of four threonine residues were found to be involved in the interactions with conservative motifs important for the enzyme activity. Two of the threonine residues (T167 and T165) are involved in ionic interactions with an arginine cluster from αC-helix. The third residue (T163) plays a crucial role, interacting with His-Arg-Asp triad (HRD). Last of the threonine residues (T162), as well as the serine (S214), were indicated to play a role in the substrate recognition or dimerization of the enzyme. The presence of ATP in the unphosphorylated model induced conformational instability of the activation loop and Asp-Phe-Gly motif (DFG). Based on our calculations we put forward a hypothesis suggesting that the ATP binds after phosphorylation of the activation loop to create a fully active conformation in the closed position.

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Abbreviations

ATP:

Adenosine triphosphate

DFG-motif:

Asp-Phe-Gly conserved kinase motif

HRD-motif:

His-Arg-Asp conserved kinase motif

PASTA:

Penicillin-binding protein and serine/threonine kinase associated domain

PrkCc:

Catalytic domain of PrkC kinase

PrkCc-ATP:

Complex of PrkCc with ATP

pPrkCc:

Phosphorylated catalytic domain of PrkC kinase

pPrkCc-ATP:

Complex of pPrkCc with ATP

RMSd:

Root mean square deviation

RMSf:

Root mean square fluctuation

SAS:

Solvent-accessible surface

References

  1. Rubin GM, Yandell MD, Wortman JR, Gabor Miklos GL, Nelson CR, Hariharan IK, Fortini ME, Li PW, Apweiler R, Fleischmann W, Cherry JM, Henikoff S, Skupski MP, Misra S, Ashburner M, Birney E, Boguski MS, Brody T, Brokstein P, Celniker SE, Chervitz SA, Coates D, Cravchik A, Gabrielian A, Galle RF, Gelbart WM, George RA, Goldstein LS, Gong F, Guan P, Harris NL, Hay BA, Hoskins RA, Li J, Li Z, Hynes RO, Jones SJ, Kuehl PM, Lemaitre B, Littleton JT, Morrison DK, Mungall C, O’Farrell PH, Pickeral OK, Shue C, Vosshall LB, Zhang J, Zhao Q, Zheng XH and Lewis S (2000) Comparative genomics of the eukaryotes. Science 287: 2204

  2. Hakansson S, Galyov EE, Rosqvist R, Wolf-Watz H (1996) The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane. Mol Microbiol 20:593

    Article  CAS  Google Scholar 

  3. Hinc K, Nagorska K, Iwanicki A, Wegrzyn G, Seror SJ, Obuchowski M (2006) Expression of genes coding for GerA and GerK spore germination receptors is dependent on the protein phosphatase. PrpE J Bacteriol 188:4373

    Article  CAS  Google Scholar 

  4. Madec E, Laszkiewicz A, Iwanicki A, Obuchowski M, Seror S (2002) Characterization of a membrane-linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes. Mol Microbiol 46:571

    Article  CAS  Google Scholar 

  5. Petrickova K, Petricek M (2003) Eukaryotic-type protein kinases in Streptomyces coelicolor: variations on a common theme. Microbiology 149:1609

    Article  CAS  Google Scholar 

  6. Shi L, Potts M, Kennelly PJ (1998) The serine, threonine and/or tyrosine-specific protein kinases and protein phosphatases of prokaryotic organisms: a family portrait. FEMS Microbiol Rev 22:229

    Article  CAS  Google Scholar 

  7. Iwanicki A, Herman-Antosiewicz A, Pierechod M, Seror SJ, Obuchowski M (2002) PrpE, a PPP protein phosphatase from Bacillus subtilis with unusual substrate specificity. Biochem J 366:929

    CAS  Google Scholar 

  8. Obuchowski M, Madec E, Delattre D, Boel G, Iwanicki A, Foulger D, Seror SJ (2000) Characterization of PrpC from Bacillus subtilis, a member of the PPM phosphatase family. J Bacteriol 182:5634

    Article  CAS  Google Scholar 

  9. Vijay K, Brody MS, Fredlund E, Price CW (2000) A PP2C phosphatase containing a PAS domain is required to convey signals of energy stress to the sigmaB transcription factor of Bacillus subtilis. Mol Microbiol 35:180

    Article  CAS  Google Scholar 

  10. Yudkin MD, Clarkson J (2005) Differential gene expression in genetically identical sister cells: the initiation of sporulation in Bacillus subtilis. Mol Microbiol 56:578

    Article  CAS  Google Scholar 

  11. Mijakovic I, Poncet S, Boel G, Maze A, Gillet S, Jamet E, Decottignies P, Grangeasse C, Doublet P, Le Marechal P, Deutscher J (2003) Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases. EMBO J 22:4709

    Article  CAS  Google Scholar 

  12. Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM (1991) Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253:407

    Article  CAS  Google Scholar 

  13. De Bondt HL, Rosenblatt J, Jancarik J, Jones HD, Morgan DO, Kim SH (1993) Crystal structure of cyclin-dependent kinase 2. Nature 363:595

    Article  Google Scholar 

  14. Goldberg J, Nairn AC, Kuriyan J (1996) Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I. Cell 84:875

    Article  CAS  Google Scholar 

  15. Hubbard SR, Wei L, Ellis L, Hendrickson WA (1994) Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature 372:746

    Article  CAS  Google Scholar 

  16. Zhang F, Strand A, Robbins D, Cobb MH, Goldsmith EJ (1994) Atomic structure of the MAP kinase ERK2 at 2.3 A resolution. Nature 367:704

    Article  CAS  Google Scholar 

  17. Johnson LN, Lewis RJ (2001) Structural basis for control by phosphorylation. Chem Rev 101:2209

    Article  CAS  Google Scholar 

  18. Kannan N, Neuwald AF (2005) Did protein kinase regulatory mechanisms evolve through elaboration of a simple structural component? J Mol Biol 351:956

    Article  CAS  Google Scholar 

  19. Kornev AP, Haste NM, Taylor SS, Eyck LF (2006) Surface comparison of active and inactive protein kinases identifies a conserved activation mechanism. Proc Natl Acad Sci USA 103:17783

    Article  CAS  Google Scholar 

  20. Groban ES, Narayanan A, Jacobson MP (2006) Conformational changes in protein loops and helices induced by post-translational phosphorylation. PLoS Comput Biol 2:e32

    Article  Google Scholar 

  21. Banavali NK, Roux B (2007) Anatomy of a structural pathway for activation of the catalytic domain of Src kinase Hck. Proteins 67:1096

    Article  CAS  Google Scholar 

  22. Shah IM, Laaberki MH, Popham DL, Dworkin J (2008) A eukaryotic-like Ser/Thr kinase signals bacteria to exit dormancy in response to peptidoglycan fragments. Cell 135:486

    Article  CAS  Google Scholar 

  23. Gerhardt P, Marquis RE (1989) Spore thermoresistance mechanisms. In: Smith I, Slepecky RA, Setlow P (eds) Regulation of Prokaryotic Development American Society for Microbiology, Washington, DC, pp 43–63

  24. Kazakov S, Bonvouloir E, Gazaryan I (2008) Physicochemical characterization of natural ionic Microreservoirs: Bacillus subtilis dormant spores. J Phys Chem B 112:2233

    Article  CAS  Google Scholar 

  25. Setlow P, Kornberg A (1970) Biochemical studies of bacterial sporulation and germination. XXII. Energy metabolism in early stages of germination of Bacillus megaterium spores. J Biol Chem 245:3637

    CAS  Google Scholar 

  26. Madec E, Stensballe A, Kjellstrom S, Cladiere L, Obuchowski M, Jensen ON, Seror SJ (2003) Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis. J Mol Biol 330:459

    Article  CAS  Google Scholar 

  27. Johnson LN, Noble ME, Owen DJ (1996) Active and inactive protein kinases: structural basis for regulation. Cell 85:149

    Article  CAS  Google Scholar 

  28. Wehenkel A, Fernandez P, Bellinzoni M, Catherinot V, Barilone N, Labesse G, Jackson M, Alzari PM (2006) The structure of PknB in complex with mitoxantrone, an ATP-competitive inhibitor, suggests a mode of protein kinase regulation in mycobacteria. FEBS Lett 580:3018

    Article  CAS  Google Scholar 

  29. Fernandez P, Saint-Joanis B, Barilone N, Jackson M, Gicquel B, Cole ST, Alzari PM (2006) The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth. J Bacteriol 188:7778

    Article  CAS  Google Scholar 

  30. Duran R, Villarino A, Bellinzoni M, Wehenkel A, Fernandez P, Boitel B, Cole ST, Alzari PM, Cervenansky C (2005) Conserved autophosphorylation pattern in activation loops and juxtamembrane regions of Mycobacterium tuberculosis Ser/Thr protein kinases. Biochem Biophys Res Commun 333:858

    Article  CAS  Google Scholar 

  31. Boitel B, Ortiz-Lombardia M, Duran R, Pompeo F, Cole ST, Cervenansky C, Alzari PM (2003) PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis. Mol Microbiol 49:1493

    Article  CAS  Google Scholar 

  32. Vulpetti A, Bosotti R (2004) Sequence and structural analysis of kinase ATP pocket residues. Farmaco 59:759

    Article  CAS  Google Scholar 

  33. Pedretti A, Villa L, Vistoli G (2002) VEGA: a versatile program to convert, handle and visualize molecular structure on Windows-based PCs. J Mol Graph Model 21:47

    Article  CAS  Google Scholar 

  34. Adams JA (2001) Kinetic and catalytic mechanisms of protein kinases. Chem Rev 101:2271

    Article  CAS  Google Scholar 

  35. Krupa A, Preethi G, Srinivasan N (2004) Structural modes of stabilization of permissive phosphorylation sites in protein kinases: distinct strategies in Ser/Thr and Tyr kinases. J Mol Biol 339:1025

    Article  CAS  Google Scholar 

  36. Gay LM, Ng HL, Alber T (2006) A conserved dimer and global conformational changes in the structure of apo-PknE Ser/Thr protein kinase from Mycobacterium tuberculosis. J Mol Biol 360:409

    Article  CAS  Google Scholar 

  37. Nolen B, Taylor S, Ghosh G (2004) Regulation of protein kinases; controlling activity through activation segment conformation. Mol Cell 15:661

    Article  CAS  Google Scholar 

  38. Shan Y, Seeliger MA, Eastwood MP, Frank F, Xu H, Jensen MO, Dror RO, Kuriyan J, Shaw DE (2009) A conserved protonation-dependent switch controls drug binding in the Abl kinase. Proc Natl Acad Sci USA 106:139

    Article  CAS  Google Scholar 

  39. Zheng J, Knighton DR, ten Eyck LF, Karlsson R, Xuong N, Taylor SS, Sowadski JM (1993) Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry 32:2154

    Article  CAS  Google Scholar 

  40. Bossemeyer D, Engh RA, Kinzel V, Ponstingl H, Huber R (1993) Phosphotransferase and substrate binding mechanism of the cAMP-dependent protein kinase catalytic subunit from porcine heart as deduced from the 2.0 A structure of the complex with Mn2 + adenylyl imidodiphosphate and inhibitor peptide PKI(5–24). EMBO J 12:849

    CAS  Google Scholar 

  41. Noble ME, Endicott JA, Johnson LN (2004) Protein kinase inhibitors: insights into drug design from structure. Science 303:1800

    Article  CAS  Google Scholar 

  42. Liu Y, Gray NS (2006) Rational design of inhibitors that bind to inactive kinase conformations. Nat Chem Biol 2:358

    Article  CAS  Google Scholar 

  43. Xu W, Harrison SC, Eck MJ (1997) Three-dimensional structure of the tyrosine kinase c-Src. Nature 385:595

    Article  CAS  Google Scholar 

  44. Sicheri F, Moarefi I, Kuriyan J (1997) Crystal structure of the Src family tyrosine kinase Hck. Nature 385:602

    Article  CAS  Google Scholar 

  45. Wood ER, Truesdale AT, McDonald OB, Yuan D, Hassell A, Dickerson SH, Ellis B, Pennisi C, Horne E, Lackey K, Alligood KJ, Rusnak DW, Gilmer TM, Shewchuk L (2004) A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res 64:6652

    Article  CAS  Google Scholar 

  46. Sheridan RP, Holloway MK, McGaughey G, Mosley RT, Singh SB (2002) A simple method for visualizing the differences between related receptor sites. J Mol Graph Model 21:217

    Article  Google Scholar 

  47. Zhou J, Adams JA (1997) Is there a catalytic base in the active site of cAMP-dependent protein kinase? Biochemistry 36:2977

    Article  CAS  Google Scholar 

  48. Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27:157

    Article  CAS  Google Scholar 

  49. Ortiz-Lombardia M, Pompeo F, Boitel B, Alzari PM (2003) Crystal structure of the catalytic domain of the PknB serine/threonine kinase from Mycobacterium tuberculosis. J Biol Chem 278:13094

    Article  CAS  Google Scholar 

  50. Madhusudan AkamineP, Xuong NH, Taylor SS (2002) Crystal structure of a transition state mimic of the catalytic subunit of cAMP-dependent protein kinase. Nat Struct Biol 9:273

    Article  CAS  Google Scholar 

  51. Cox S, Taylor SS (1995) Kinetic analysis of cAMP-dependent protein kinase: mutations at histidine 87 affect peptide binding and pH dependence. Biochemistry 34:16203

    Article  CAS  Google Scholar 

  52. Cole JL (2007) Activation of PKR: an open and shut case? Trends Biochem Sci 32:57

    Article  CAS  Google Scholar 

  53. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381

    Article  CAS  Google Scholar 

  54. Wang J, Cieplak P, Kollman P (2000) How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J Comput Chem 21:1049

    Article  CAS  Google Scholar 

  55. Gruszczyński P, Kaźmierkiewicz R, Obuchowski M, Lammek B (2007) Theoretical modeling of PrkCc, serine-threonine protein kinase intracellular domain, complexed with ATP derivatives QSAR. Comb Sci 27:437

    Article  Google Scholar 

  56. Case DA, Cheatham TE 3rd, Darden T, Gohlke H, Luo R, Merz KM Jr, Onufriev A, Simmerling C, Wang B, Woods RJ (2005) The Amber biomolecular simulation programs. J Comput Chem 26:1668

    Article  CAS  Google Scholar 

  57. Homeyer N, Horn AH, Lanig H, Sticht H (2006) AMBER force-field parameters for phosphorylated amino acids in different protonation states: phosphoserine, phosphothreonine, phosphotyrosine, and phosphohistidine. J Mol Model 12:281

    Article  CAS  Google Scholar 

  58. Narayanan A, Jacobson MP (2009) Computational studies of protein regulation by post-translational phosphorylation. Curr Opin Struct Biol 19:156

    Article  CAS  Google Scholar 

  59. Sugawara Y, Iwasaki H (1984) Crystal transformation and conformational change of disodium adenosine 5’-triphosphate and the structure of ATP Na2·2H2O. Acta Crystallogr A 40:C68

    Article  Google Scholar 

  60. Schaftenaar G, Noordik JH (2000) Molden: a pre- and post-processing program for molecular and electronic structures. J Comput Aided Mol Des 14:123

    Article  CAS  Google Scholar 

  61. Schmidt M, Baldridge K, Boatz J, Elbert S, Gordon M, Jensen J, Koseki S, Matsunaga N, Nguyen K, Su S, Windus T, Dupuis M, Montgomery J (1993) General atomic and molecular electronic structure system. J Comput Chem 14:1347

    Article  CAS  Google Scholar 

  62. Bayly CI, Cieplak P, Cornell WD, Kollman PA (1993) A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J Phys Chem 97:10269

    Article  CAS  Google Scholar 

  63. Meagher K, Redman L, Carlson H (2003) Development of polyphosphate parameters for use with the AMBER force field. J Comput Chem 24:1016

    Article  CAS  Google Scholar 

  64. Morris G, Goodsell D, Halliday R, Huey R, Hart W, Belew R, Olson A (1999) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 19:1639

    Article  Google Scholar 

  65. Holland J (1992) Adaptation in natural and artificial systems: an introductory analysis with applications to biology, control and artificial intelligence. The MIT Press, Cambridge

    Google Scholar 

  66. Sayle RA, Milner-White EJ (1995) RASMOL: biomolecular graphics for all trends. Biochem Sci 20:374

    Article  CAS  Google Scholar 

  67. Ryckaert J-P, Ciccotti G, Berendsen H (1977) Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comput Phys 23:327

    Article  CAS  Google Scholar 

  68. Miyamoto S, Kollman P (1992) SETTLE: an analytical version of the SHAKE and RATTLE algorithm for rigid water models. J Comput Chem 13:952

    Article  CAS  Google Scholar 

  69. Berendsen HJC, Postma JPM, Vangunsteren WF, Dinola A, Haak JR (1984) Molecular-dynamics with coupling to an external bath. J Chem Phys 81:3684

    Article  CAS  Google Scholar 

  70. Delano WL (2002) The PyMOL molecular graphics system on World Wide Web http://www.pymol.org

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Acknowledgments

We acknowledge Tiffany S. Han for critical reading of the manuscript. This work was partially supported by University of Gdańsk fund DS/B50A-4-162-8.

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Authors and Affiliations

  1. Faculty of Chemistry, University of Gdańsk, Sobieskiego 18/19, 80-952, Gdańsk, Poland

    Paweł Gruszczyński

  2. Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Kładki 24, 80-822, Gdańsk, Poland

    Paweł Gruszczyński & Rajmund Kaźmierkiewicz

  3. Department of Medical Biotechnology, Intercollegiate Faculty of Biotechnology, Medical University of Gdańsk, Dębinki 1, 80-811, Gdańsk, Poland

    Michał Obuchowski

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  1. Paweł Gruszczyński
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Correspondence to Paweł Gruszczyński.

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Gruszczyński, P., Obuchowski, M. & Kaźmierkiewicz, R. Phosphorylation and ATP-binding induced conformational changes in the PrkC, Ser/Thr kinase from B. subtilis . J Comput Aided Mol Des 24, 733–747 (2010). https://doi.org/10.1007/s10822-010-9370-4

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