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Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases.

Wehenkel A, Bellinzoni M, Schaeffer F, Villarino A, Alzari PM
J Mol Biol 374 890-8 (2007)
Cited: 42 times
EuropePMC logo PMID: 17961594

Abstract

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the alpha isoform of human PP2C (PP2Calpha), which folds into an alpha/beta domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn(2+)-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.

Articles - 2cm1 mentioned but not cited (2)

  1. Binding of a third metal ion by the human phosphatases PP2Cα and Wip1 is required for phosphatase activity. Tanoue K, Miller Jenkins LM, Durell SR, Debnath S, Sakai H, Tagad HD, Ishida K, Appella E, Mazur SJ. Biochemistry 52 5830-5843 (2013)
  2. Mycobacterial phosphatase PstP regulates global serine threonine phosphorylation and cell division. Iswahyudi, Mukamolova GV, Straatman-Iwanowska AA, Allcock N, Ajuh P, Turapov O, O'Hare HM. Sci Rep 9 8337 (2019)


Reviews citing this publication (8)

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  8. Targeting the messengers: Serine/threonine protein kinases as potential targets for antimycobacterial drug development. Khan MZ, Kaur P, Nandicoori VK. IUBMB Life 70 889-904 (2018)

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  11. Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen. Sharma AK, Arora D, Singh LK, Gangwal A, Sajid A, Molle V, Singh Y, Nandicoori VK. J Biol Chem 291 24215-24230 (2016)
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  13. Optimization of a cyclic peptide inhibitor of Ser/Thr phosphatase PPM1D (Wip1). Hayashi R, Tanoue K, Durell SR, Chatterjee DK, Jenkins LM, Appella DH, Appella E. Biochemistry 50 4537-4549 (2011)
  14. Bypass suppression analysis maps the signalling pathway within a multidomain protein: the RsbP energy stress phosphatase 2C from Bacillus subtilis. Brody MS, Stewart V, Price CW. Mol Microbiol 72 1221-1234 (2009)
  15. Structure of the phosphatase domain of the cell fate determinant SpoIIE from Bacillus subtilis. Levdikov VM, Blagova EV, Rawlings AE, Jameson K, Tunaley J, Hart DJ, Barak I, Wilkinson AJ. J Mol Biol 415 343-358 (2012)
  16. Determinants for substrate specificity of the bacterial PP2C protein phosphatase tPphA from Thermosynechococcus elongatus. Su J, Forchhammer K. FEBS J 280 694-707 (2013)
  17. Structural Mechanism Underlying the Specific Recognition between the Arabidopsis State-Transition Phosphatase TAP38/PPH1 and Phosphorylated Light-Harvesting Complex Protein Lhcb1. Wei X, Guo J, Li M, Liu Z. Plant Cell 27 1113-1127 (2015)
  18. The UDP-diacylglucosamine pyrophosphohydrolase LpxH in lipid A biosynthesis utilizes Mn2+ cluster for catalysis. Young HE, Donohue MP, Smirnova TI, Smirnov AI, Zhou P. J Biol Chem 288 26987-27001 (2013)
  19. 5,5'-Methylenedisalicylic Acid (MDSA) Modulates SarA/MgrA Phosphorylation by Targeting Ser/Thr Phosphatase Stp1. Zheng W, Liang Y, Zhao H, Zhang J, Li Z. Chembiochem 16 1035-1040 (2015)
  20. Identification and modelling of a PPM protein phosphatase fold in the Legionella pneumophila deAMPylase SidD. Rigden DJ. FEBS Lett 585 2749-2754 (2011)
  21. Substrate-dependent metal preference of PPM1H, a cancer-associated protein phosphatase 2C: comparison with other family members. Sugiura T, Noguchi Y. Biometals 22 469-477 (2009)
  22. Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry. Mazur SJ, Gallagher ES, Debnath S, Durell SR, Anderson KW, Miller Jenkins LM, Appella E, Hudgens JW. Biochemistry 56 2676-2689 (2017)
  23. Structure of the RsbX phosphatase involved in the general stress response of Bacillus subtilis. Teh AH, Makino M, Hoshino T, Baba S, Shimizu N, Yamamoto M, Kumasaka T. Acta Crystallogr D Biol Crystallogr 71 1392-1399 (2015)
  24. A PPM-family protein phosphatase from the thermoacidophile Thermoplasma volcanium hydrolyzes protein-bound phosphotyrosine. Dahche H, Abdullah A, Ben Potters M, Kennelly PJ. Extremophiles 13 371-377 (2009)
  25. Crystal structure and catalytic activity of the PPM1K N94K mutant. Dolatabad MR, Guo LL, Xiao P, Zhu Z, He QT, Yang DX, Qu CX, Guo SC, Fu XL, Li RR, Ge L, Hu KJ, Liu HD, Shen YM, Yu X, Sun JP, Zhang PJ. J Neurochem 148 550-560 (2019)
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  28. The Role of Arg13 in Protein Phosphatase M tPphA from Thermosynechococcus elongatus. Su J, Forchhammer K. Enzyme Res 2012 272706 (2012)
  29. The inhibitory mechanism of aurintricarboxylic acid targeting serine/threonine phosphatase Stp1 in Staphylococcus aureus: insights from molecular dynamics simulations. Liu TT, Yang T, Gao MN, Chen KX, Yang S, Yu KQ, Jiang HL. Acta Pharmacol Sin 40 850-858 (2019)
  30. Rv2577 of Mycobacterium tuberculosis Is a Virulence Factor With Dual Phosphatase and Phosphodiesterase Functions. Forrellad MA, Blanco FC, Marrero Diaz de Villegas R, Vázquez CL, Yaneff A, García EA, Gutierrez MG, Durán R, Villarino A, Bigi F. Front Microbiol 11 570794 (2020)
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