Skip to main content
SpringerLink
Log in

The Role of Strong Electrostatic Interactions at the Dimer Interface of Human Glutathione Synthetase

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

The obligate homodimer human glutathione synthetase (hGS) provides an ideal system for exploring the role of protein–protein interactions in the structural stability, activity and allostery of enzymes. The two active sites of hGS, which are 40 Å apart, display allosteric modulation by the substrate γ-glutamylcysteine (γ-GC) during the synthesis of glutathione, a key cellular antioxidant. The two subunits interact at a relatively small dimer interface dominated by electrostatic interactions between S42, R221, and D24. Alanine scans of these sites result in enzymes with decreased activity, altered γ-GC affinity, and decreased thermal stability. Molecular dynamics simulations indicate these mutations disrupt interchain bonding and impact the tertiary structure of hGS. While the ionic hydrogen bonds and salt bridges between S42, R221, and D24 do not mediate allosteric communication in hGS, these interactions have a dramatic impact on the activity and structural stability of the enzyme.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

hGS:

Human glutathione synthetase

GSH:

Glutathione

γ-GC:

γ-Glutamylcysteine

γ-GluABA:

l-γ-Glutamyl-l-α-aminobutyrate

IPTG:

Isopropyl-1-thio-β-galactopyranoside

PK:

Pyruvate kinase

LDH:

Lactate dehydrogenase

DSC:

Differential scanning calorimetry

2HGS:

Crystal structure of human glutathione synthetase

WT:

Wild-type

Tm :

Transition midpoint

MD:

Molecular dynamics

RMSD:

Root mean square deviation

References

  1. Haber JE, Koshland DE Jr (1967) Relation of protein subunit interactions to the molecular species observed during cooperative binding of ligands. Proc Natl Acad Sci USA 58:2087–2093

    Article  CAS  Google Scholar 

  2. Kirtley ME, Koshland DE Jr (1967) Models for cooperative effects in proteins containing subunits. Effects of two interacting ligands. J Biol Chem 242:4192–4205

    CAS  Google Scholar 

  3. Kantrowitz ER (2012) Allostery and cooperativity in Escherichia coli aspartate transcarbamoylase. Arch Biochem Biophys 519:81–90

    Article  CAS  Google Scholar 

  4. May LT, Leach K, Sexton PM, Christopoulos A (2007) Allosteric modulation of G-protein coupled receptors. Annu Rev Pharmacol Toxicol 47:1–51

    Article  CAS  Google Scholar 

  5. Geitmann M, Elinder M, Seeger C, Brandt P, de Esch IWJ, Danielson UH (2011) Identification of a novel scaffold for allosteric inhibition of wild type and drug resistant HIV-1 reverse transcriptase by fragment library screening. J Med Chem 54:699–708

    Article  CAS  Google Scholar 

  6. Wells JA, McClendon CL (2007) Reaching for high hanging fruit in drug discovery at protein–protein interfaces. Nature 450:1001–1009

    Article  CAS  Google Scholar 

  7. Bohr C, Hasselbach KA, Krogh A (1904) Skan. Arch Physiol 16:402–412 quoted in Koshland DE Jr, Hamadani K (2002) Proteomics and models for enzyme cooperativity. J Biol Chem 277:46841–46844

  8. Kalodimos CG (2012) Protein function and allostery: a dynamic relationship. Ann NY Acad Sci 1260:81–86

    Article  CAS  Google Scholar 

  9. Rader AJ, Brown SM (2011) Correlating allostery with rigidity. Mol BioSyst 7:464–471

    Article  CAS  Google Scholar 

  10. Jones S, Thornton JM (1996) Principles of protein–protein interactions. Pro Natl Acad Sci USA 93:13–20

    Article  CAS  Google Scholar 

  11. Keskin O, Gursoy A, Ma B, Nussinov R (2008) Principles of protein–protein interactions: what are the preferred ways for proteins to interact? Chem Rev 108:1225–1244

    Article  CAS  Google Scholar 

  12. Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760

    Article  CAS  Google Scholar 

  13. Oppenheimer L, Wellner VP, Griffith OW, Meister A (1979) Glutathione synthetase purification from rat kidney and mapping of the substrate binding sites. J Biol Chem 254:5184–5189

    CAS  Google Scholar 

  14. Bush EC, Clark AE, DeBoever CM, Haynes LE, Hussain S, Ma S, McDermott MM, Novak AM, Wentworth JS (2012) Modeling the role of negative cooperativity in metabolic regulation and homeostasis. PLoS ONE 7:e48920

    Article  CAS  Google Scholar 

  15. Cornish-Bowden A (2013) The physiological significance of negative cooperativity revisited. J Theor Biol 319:144–147

    Article  Google Scholar 

  16. Galperin MY, Koonin EV (1997) A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity. Protein Sci 6:2639–2643

    Article  CAS  Google Scholar 

  17. Fawaz MV, Topper ME, Firestine SM (2011) The ATP-grasp enzymes. Bioorg Chem 39:185–191

    Article  CAS  Google Scholar 

  18. Ristoff R, Larsson A (2002) Oxidative stress in inborn errors of metabolism: lessons from glutathione deficiency. J Inherit Metab Dis 25:223–226

    Article  CAS  Google Scholar 

  19. Bains JS, Shaw CA (1997) Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death. Brain Res Rev 25:335–358

    Article  CAS  Google Scholar 

  20. Larsson A, Ristoff E, Anderson ME (2005) Glutathione synthetase deficiency and other disorders of the γ-glutamyl cycle. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) Metabolic basis of inherited disease, Online (genetics.accessmedicine.com). McGraw Hill, New York

  21. Townsend DM, Tew KD, Tapiero H (2003) The importance of glutathione in human disease. Biomed Pharmacother 57:145–155

    Article  CAS  Google Scholar 

  22. Slavens KD, Brown TR, Barakat KA, Cundari TR, Anderson ME (2011) Valine 44 and valine 45 of human glutathione synthetase are key for subunit stability and negative cooperativity. Biochem Biophys Res Commun 410:597–601

    Article  CAS  Google Scholar 

  23. Dinescu A, Cundari TR, Bhansali VS, Luo JL, Anderson ME (2004) Function of conserved residues of human glutathione synthetase. J Biol Chem 279:22412–22421

    Article  CAS  Google Scholar 

  24. Dinescu A, Brown TR, Barelier S, Cundari TR, Anderson ME (2010) The role of the glycine triad in human glutathione synthetase. Biochem Biophys Res Commun 400:511–516

    Article  CAS  Google Scholar 

  25. Brown TR, Drummond ML, Barelier S, Crutchfield AS, Dinescu A, Slavens KD, Cundari TR, Anderson ME (2011) Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure. Biochem Biophys Res Comm 411:536–542

    Article  CAS  Google Scholar 

  26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  27. Luo JL, Huang CS, Babaoglu K, Anderson ME (2000) Novel kinetics of mammalian glutathione synthetase: characterization of gamma-glutamyl substrate cooperative binding. Biochem Biophys Res Commun 275:577–581

    Article  CAS  Google Scholar 

  28. Dinescu A, Anderson ME, Cundari TR (2007) Catalytic loop motion in human glutathione synthetase: a molecular modeling approach. Biochem Biophys Res Commun 353:450–456

    Article  CAS  Google Scholar 

  29. Polekhina G, Board PG, Gali RR, Rossjohn J, Parker MW (1999) Molecular basis of glutathione synthetase deficiency and a rare gene permutation event. EMBO J 12:3204–3213

    Article  Google Scholar 

Download references

Acknowledgments

The authors thank the Chemical Computing Group for providing the MOE software. We also thank Amy Graves, Teresa Brown, and Sarah Barelier for technical assistance and Mark Britt and Richard Sheardy for instrumentation assistance. Supported in part by NIH R15GM086833 (MEA), a Research Enhancement Program Grant (TWU, MEA), and a UNT Faculty Research Grant (TRC).

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Texas Woman’s University, P.O. Box 425859, Denton, TX, 76204, USA

    Margarita C. De Jesus, Bisesh Shrestha, Kerri D. Slavens & Mary E. Anderson

  2. Center for Advanced Scientific Computing and Modeling (CASCaM), Department of Chemistry, University of North Texas, Denton, TX, 76203, USA

    Brandall L. Ingle, Khaldoon A. Barakat & Thomas R. Cundari

Authors
  1. Margarita C. De Jesus
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brandall L. Ingle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Khaldoon A. Barakat
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bisesh Shrestha
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kerri D. Slavens
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Thomas R. Cundari
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mary E. Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mary E. Anderson.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 1,428 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

De Jesus, M.C., Ingle, B.L., Barakat, K.A. et al. The Role of Strong Electrostatic Interactions at the Dimer Interface of Human Glutathione Synthetase. Protein J 33, 403–409 (2014). https://doi.org/10.1007/s10930-014-9573-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-014-9573-y

Keywords

Search

Navigation