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Contribution of glycine 146 to a conserved folding module affecting stability and refolding of human glutathione transferase p1-1.

Kong GK, Polekhina G, McKinstry WJ, Parker MW, Dragani B, Aceto A, Paludi D, Principe DR, Mannervik B, Stenberg G
J Biol Chem 278 1291-302 (2003)
Cited: 16 times
EuropePMC logo PMID: 12414796

Abstract

In human glutathione transferase P1-1 (hGSTP1-1) position 146 is occupied by a glycine residue, which is located in a bend of a long loop that together with the alpha6-helix forms a substructure (GST motif II) maintained in all soluble GSTs. In the present study G146A and G146V mutants were generated by site-directed mutagenesis in order to investigate the function played by this conserved residue in folding and stability of hGSTP1-1. Crystallographic analysis of the G146V variant, expressed at the permissive temperature of 25 degrees C, indicates that the mutation causes a substantial change of the backbone conformation because of steric hindrance. Stability measurements indicate that this mutant is inactivated at a temperature as low as 32 degrees C. The structure of the G146A mutant is identical to that of the wild type with the mutated residue having main-chain bond angles in a high energy region of the Ramachandran plot. However even this Gly --> Ala substitution inactivates the enzyme at 37 degrees C. Thermodynamic analysis of all variants confirms, together with previous findings, the critical role played by GST motif II for overall protein stability. Analysis of reactivation in vitro indicates that any mutation of Gly-146 alters the folding pathway by favoring aggregation at 37 degrees C. It is hypothesized that the GST motif II is involved in the nucleation mechanism of the protein and that the substitution of Gly-146 alters this transient substructure. Gly-146 is part of the buried local sequence GXXh(T/S)XXDh (X is any residue and h is a hydrophobic residue), conserved in all GSTs and related proteins that seems to behave as a characteristic structural module important for protein folding and stability.

Reviews citing this publication (3)

  1. Insect glutathione transferases. Ketterman AJ, Saisawang C, Wongsantichon J. Drug Metab Rev 43 253-265 (2011)
  2. The still mysterious roles of cysteine-containing glutathione transferases in plants. Lallement PA, Brouwer B, Keech O, Hecker A, Rouhier N. Front Pharmacol 5 192 (2014)
  3. Glyphosate's Synergistic Toxicity in Combination with Other Factors as a Cause of Chronic Kidney Disease of Unknown Origin. Gunatilake S, Seneff S, Orlando L. Int J Environ Res Public Health 16 E2734 (2019)

Articles citing this publication (13)

  1. Recombinant protein glutathione S-transferases P1 attenuates inflammation in mice. Luo L, Wang Y, Feng Q, Zhang H, Xue B, Shen J, Ye Y, Han X, Ma H, Xu J, Chen D, Yin Z. Mol Immunol 46 848-857 (2009)
  2. Identification of a conserved N-capping box important for the structural autonomy of the prion alpha 3-helix: the disease associated D202N mutation destabilizes the helical conformation. Gallo M, Paludi D, Cicero DO, Chiovitti K, Millo E, Salis A, Damonte G, Corsaro A, Thellung S, Schettini G, Melino S, Florio T, Paci M, Aceto A. Int J Immunopathol Pharmacol 18 95-112 (2005)
  3. Evolutionarily conserved structural motifs in bacterial GST (glutathione S-transferase) are involved in protein folding and stability. Allocati N, Masulli M, Pietracupa M, Federici L, Di Ilio C. Biochem J 394 11-17 (2006)
  4. Cloning, characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[alpha]pyrene exposure. Xu C, Pan L, Liu N, Wang L, Miao J. Comp Biochem Physiol C Toxicol Pharmacol 152 160-166 (2010)
  5. Glutamate-64, a newly identified residue of the functionally conserved electron-sharing network contributes to catalysis and structural integrity of glutathione transferases. Winayanuwattikun P, Ketterman AJ. Biochem J 402 339-348 (2007)
  6. Conserved glycines in the C terminus of MinC proteins are implicated in their functionality as cell division inhibitors. Ramirez-Arcos S, Greco V, Douglas H, Tessier D, Fan D, Szeto J, Wang J, Dillon JR. J Bacteriol 186 2841-2855 (2004)
  7. A sensitive core region in the structure of glutathione S-transferases. Wongsantichon J, Harnnoi T, Ketterman AJ. Biochem J 373 759-765 (2003)
  8. Molecular identification of glutathione S-transferase gene and cDNAs of two isotypes from northern quahog (Mercenaria mercenaria). Feng X, Singh BR. Comp Biochem Physiol B Biochem Mol Biol 154 25-36 (2009)
  9. Expression, purification and functional analysis of hexahistidine-tagged human glutathione S-transferase P1-1 and its cysteinyl mutants. Wu Y, Shen J, Yin Z. Protein J 26 359-370 (2007)
  10. Functional studies of single-nucleotide polymorphic variants of human glutathione transferase T1-1 involving residues in the dimer interface. Josephy PD, Pan D, Ianni MD, Mannervik B. Arch Biochem Biophys 513 87-93 (2011)
  11. Intra-subunit residue interactions from the protein surface to the active site of glutathione S-transferase AdGSTD3-3 impact on structure and enzyme properties. Wongtrakul J, Wongtrakul J, Sramala I, Prapanthadara LA, Ketterman AJ. Insect Biochem Mol Biol 35 197-205 (2005)
  12. The conserved glycine/alanine residue of the active-site loop containing the putative acetylCoA-binding motif is essential for the overall structural integrity of Mesorhizobium loti arylamine N-acetyltransferase 1. Atmane N, Dairou J, Flatters D, Martins M, Pluvinage B, Derreumaux P, Dupret JM, Rodrigues-Lima F. Biochem Biophys Res Commun 361 256-262 (2007)
  13. Structural assessment of glycyl mutations in invariantly conserved motifs. Prakash T, Sandhu KS, Singh NK, Bhasin Y, Ramakrishnan C, Brahmachari SK. Proteins 69 617-632 (2007)


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