Energetics of Glutathione Binding to Human Eukaryotic Elongation Factor 1 Gamma: Isothermal Titration Calorimetry and Molecular Dynamics Studies

Abstract

The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K = 115 μM), with more hydrogen-bond contacts (ΔH = -73.8 kJ/mol) and unfavourable entropy (-TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.

Oxidized glutathione promotes association between eukaryotic translation elongation factor 1Bγ and Ure2p glutathione transferase from Phanerochaete chrysosporium

“…The Gibbs' free energies of the different interactions (DG = ca. À5.6 kcalÁmol À1 ; [29]) are similar to those determined for PcEF1Bc (Table 3). However, contrary to PcEF1Bc, the human protein has slightly lower affinities for GSH than for GSSG (K d of 67 and 115 µM, respectively; [29]) (Table 3).…”

“…À5.6 kcalÁmol À1 ; [29]) are similar to those determined for PcEF1Bc (Table 3). However, contrary to PcEF1Bc, the human protein has slightly lower affinities for GSH than for GSSG (K d of 67 and 115 µM, respectively; [29]) (Table 3). The preference for GSH over GSSG by the full-length heEF1c could indicate that the C-terminal part may structurally influence glutathione binding to heEF1c by hindering the binding of GSSG [29].…”

“…However, contrary to PcEF1Bc, the human protein has slightly lower affinities for GSH than for GSSG (K d of 67 and 115 µM, respectively; [29]) (Table 3). The preference for GSH over GSSG by the full-length heEF1c could indicate that the C-terminal part may structurally influence glutathione binding to heEF1c by hindering the binding of GSSG [29]. Our data showed that this was not the case for PcEF1Bc, which preferentially bound GSSG over GSH, even more efficiently than the PcEF1Bc_GST domain devoid of the C-terminal part.…”

“…In human, eEF1c is able to bind GSH, GSSG, Shexylglutathione, and glutathione sulfonate, as shown by isothermal titration calorimetry (ITC) thermodynamic approaches [29]. The Gibbs' free energies of the different interactions (DG = ca.…”

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma

“…In our previous study, we showed that heEF1γ displays affinity for glutathione; hence, heEF1γ was purified by using glutathione‐Agarose affinity chromatography . We also showed that NT‐heEF1γ exhibits ligandin function toward glutathione and analogues of glutathione by using isothermal titration calorimetry . In this study, we observed that NT‐heEF1γ undergoes glutathione‐induced dimerization.…”