Crystal Structures of the Carboxyl cGMP Binding Domain of the cGMP-dependent Protein Kinase Reveal a Novel Capping Triad Crucial for Merozoite Egress

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Malaria causes up to a million fatalities per year worldwide. Most of these deaths are caused by Plasmodium falciparum, which has a complex life cycle in both humans and mosquitoes. One key regulator of this process is P. falciparum cGMP-dependent protein kinase (PfPKG), the main effector of the cGMP-signaling pathway. Specifically blocking this kinase stops both replication and transmission of the parasites, suggesting that PfPKG is a promising drug target. Here we identified the carboxyl cGMP-binding domain of PfPKG serving as a gatekeeper for activation of the entire kinase by having the highest affinity and selectivity for cGMP. High-resolution crystal structures with and without cGMP allowed us to identify a novel cGMP capping triad that dynamically forms upon binding cGMP and stabilizes the activated conformation. Mutation of the capping triad forming residues not only reduces its kinase activity, but also prevents blood stage merozoite egress, demonstrating its crucial role in PfPKG activation.

Vyšlo v časopise: Crystal Structures of the Carboxyl cGMP Binding Domain of the cGMP-dependent Protein Kinase Reveal a Novel Capping Triad Crucial for Merozoite Egress. PLoS Pathog 11(2): e32767. doi:10.1371/journal.ppat.1004639
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004639

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Zdroje

1. Cowman AF, Crabb BS (2006) Invasion of red blood cells by malaria parasites. Cell 124: 755–766. 16497586

2. Guttery DS, Holder AA, Tewari R (2012) Sexual development in Plasmodium: lessons from functional analyses. PLoS Pathog 8: e1002404. doi: 10.1371/journal.ppat.1002404 22275863

3. Doerig C, Abdi A, Bland N, Eschenlauer S, Dorin-Semblat D, et al. (2010) Malaria: targeting parasite and host cell kinomes. Biochim Biophys Acta 1804: 604–612. doi: 10.1016/j.bbapap.2009.10.009 19840874

4. Baker DA (2011) Cyclic nucleotide signalling in malaria parasites. Cell Microbiol 13: 331–339. doi: 10.1111/j.1462-5822.2010.01561.x 21176056

5. Hopp CS, Bowyer PW, Baker DA (2012) The role of cGMP signalling in regulating life cycle progression of Plasmodium. Microbes Infect 14: 831–837. doi: 10.1016/j.micinf.2012.04.011 22613210

6. McRobert L, Taylor CJ, Deng W, Fivelman QL, Cummings RM, et al. (2008) Gametogenesis in malaria parasites is mediated by the cGMP-dependent protein kinase. PLoS Biol 6: e139. doi: 10.1371/journal.pbio.0060139 18532880

7. Taylor HM, McRobert L, Grainger M, Sicard A, Dluzewski AR, et al. (2010) The malaria parasite cyclic GMP-dependent protein kinase plays a central role in blood-stage schizogony. Eukaryot Cell 9: 37–45. doi: 10.1128/EC.00186-09 19915077

8. Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, et al. (2013) Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog 9: e1003344. doi: 10.1371/journal.ppat.1003344 23675297

9. Diaz CA, Allocco J, Powles MA, Yeung L, Donald RG, et al. (2006) Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): antiparasitic activity of a PKG inhibitor. Mol Biochem Parasitol 146: 78–88. 16325279

10. Baker DA, Deng W (2005) Cyclic GMP-dependent protein kinases in protozoa. Front Biosci 10: 1229–1238. 15769621

11. Deng W, Baker DA (2002) A novel cyclic GMP-dependent protein kinase is expressed in the ring stage of the Plasmodium falciparum life cycle. Mol Microbiol 44: 1141–1151. 12068803

12. Deng W, Parbhu-Patel A, Meyer DJ, Baker DA (2003) The role of two novel regulatory sites in the activation of the cGMP-dependent protein kinase from Plasmodium falciparum. Biochem J 374: 559–565. 12817987

13. Berman HM, Ten Eyck LF, Goodsell DS, Haste NM, Kornev A, et al. (2005) The cAMP binding domain: an ancient signaling module. Proc Natl Acad Sci U S A 102: 45–50. 15618393

14. Alverdi V, Mazon H, Versluis C, Hemrika W, Esposito G, et al. (2008) cGMP-binding prepares PKG for substrate binding by disclosing the C-terminal domain. J Mol Biol 375: 1380–1393. 18082764

15. Wall ME, Francis SH, Corbin JD, Grimes K, Richie-Jannetta R, et al. (2003) Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase. Proc Natl Acad Sci U S A 100: 2380–2385. 12591946

16. Brickman E, Soll L, Beckwith J (1973) Genetic characterization of mutations which affect catabolite-sensitive operons in Escherichia coli, including deletions of the gene for adenyl cyclase. J Bacteriol 116: 582–587. 4583241

17. Osborne BW, Wu J, McFarland CJ, Nickl CK, Sankaran B, et al. (2011) Crystal structure of cGMP-dependent protein kinase reveals novel site of interchain communication. Structure 19: 1317–1327. doi: 10.1016/j.str.2011.06.012 21893290

18. Huang GY, Kim JJ, Reger AS, Lorenz R, Moon EW, et al. (2014) Structural basis for cyclic-nucleotide selectivity and cGMP-selective activation of PKG I. Structure 22: 116–124. doi: 10.1016/j.str.2013.09.021 24239458

19. Huang GY, Gerlits OO, Blakeley MP, Sankaran B, Kovalevsky AY, et al. (2014) Neutron Diffraction Reveals Hydrogen Bonds Critical for cGMP-Selective Activation: Insights for cGMP-Dependent Protein Kinase Agonist Design. Biochemistry 53: 6725–6727. doi: 10.1021/bi501012v 25271401

20. Rehmann H, Wittinghofer A, Bos JL (2007) Capturing cyclic nucleotides in action: snapshots from crystallographic studies. Nat Rev Mol Cell Biol 8: 63–73. 17183361

21. Brochet M, Collins MO, Smith TK, Thompson E, Sebastian S, et al. (2014) Phosphoinositide Metabolism Links cGMP-Dependent Protein Kinase G to Essential Ca2+ Signals at Key Decision Points in the Life Cycle of Malaria Parasites. PLoS Biol 12: e1001806. doi: 10.1371/journal.pbio.1001806 24594931

22. Adjalley SH, Lee MC, Fidock DA (2010) A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. Methods Mol Biol 634: 87–100. doi: 10.1007/978-1-60761-652-8_6 20676977

23. Kim JJ, Casteel DE, Huang G, Kwon TH, Ren RK, et al. (2011) Co-crystal structures of PKG Ibeta (92–227) with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding. PLoS One 6: e18413. doi: 10.1371/journal.pone.0018413 21526164

24. Richie-Jannetta R, Busch JL, Higgins KA, Corbin JD, Francis SH (2006) Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes. J Biol Chem 281: 6977–6984. 16407222

25. Salowe SP, Wiltsie J, Liberator PA, Donald RG (2002) The role of a parasite-specific allosteric site in the distinctive activation behavior of Eimeria tenella cGMP-dependent protein kinase. Biochemistry 41: 4385–4391. 11914085

26. Rehmann H, Prakash B, Wolf E, Rueppel A, de Rooij J, et al. (2003) Structure and regulation of the cAMP-binding domains of Epac2. Nat Struct Biol 10: 26–32. 12469113

27. Wei F, Wang W, Liu Q (2013) Protein kinases of Toxoplasma gondii: functions and drug targets. Parasitol Res 112: 2121–2129. doi: 10.1007/s00436-013-3451-y 23681193

28. Bussow K, Scheich C, Sievert V, Harttig U, Schultz J, et al. (2005) Structural genomics of human proteins—target selection and generation of a public catalogue of expression clones. Microb Cell Fact 4: 21. 15998469

29. Battye TG, Kontogiannis L, Johnson O, Powell HR, Leslie AG (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67: 271–281. doi: 10.1107/S0907444910048675 21460445

30. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, et al. (2007) Phaser crystallographic software. J Appl Crystallogr 40: 658–674. 19461840

31. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132. 15572765

32. Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, et al. (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68: 352–367. doi: 10.1107/S0907444912001308 22505256

33. Winn MD, Isupov MN, Murshudov GN (2001) Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr D Biol Crystallogr 57: 122–133. 11134934

34. Moll D, Prinz A, Gesellchen F, Drewianka S, Zimmermann B, et al. (2006) Biomolecular interaction analysis in functional proteomics. J Neural Transm 113: 1015–1032. 16835689

35. Hopp CS, Flueck C, Solyakov L, Tobin A, Baker DA (2012) Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase. PLoS One 7: e48206. doi: 10.1371/journal.pone.0048206 23139764

36. Trager W, Jensen JB (2005) Human malaria parasites in continuous culture. 1976. J Parasitol 91: 484–486. 16108535

37. Adjalley SH, Johnston GL, Li T, Eastman RT, Ekland EH, et al. (2011) Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue. Proc Natl Acad Sci U S A 108: E1214–1223. doi: 10.1073/pnas.1112037108 22042867

38. Nkrumah LJ, Muhle RA, Moura PA, Ghosh P, Hatfull GF, et al. (2006) Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nat Methods 3: 615–621. 16862136

39. Olivieri A, Collins CR, Hackett F, Withers-Martinez C, Marshall J, et al. (2011) Juxtamembrane shedding of Plasmodium falciparum AMA1 is sequence independent and essential, and helps evade invasion-inhibitory antibodies. PLoS Pathog 7: e1002448. doi: 10.1371/journal.ppat.1002448 22194692