Elsevier

Mitochondrion

Volume 46, May 2019, Pages 140-148
Mitochondrion

Adaptation of Mge1 to oxidative stress by local unfolding and altered Interaction with mitochondrial Hsp70 and Mxr2

https://doi.org/10.1016/j.mito.2018.04.003Get rights and content

Highlights

  • Oxidative stress induces local unfolding and an open conformation in Mge1.

  • Persistent oxidation causes Mge1 to form amyloid type aggregates.

  • Mxr2 acts as a chaperone to protect the oxidized Mge1 protein.

  • Altered interaction of oxidized Mge1 with Hsp70 and Mxr2.

Abstract

Perturbations in mitochondrial redox levels oxidize nucleotide exchanger Mge1, compromising its ability to bind to the Hsp70, while the Mxr2 enzyme reduces the oxidized Mge1. However, the effects of persistent oxidative stress on Mge1 structure and function are not known. In this study, we show that oxidation-induced selective and local structural adaptations cause the detachment of Mge1 from Hsp70. Notably, persistent oxidative stress causes monomeric Mge1 to aggregate and to generate amyloid-type particles. Mxr2 appears to protect Mge1 from oxidative stress induced aggregation. We conclude that the Mxr2-Mge1-Hsp70 protein triad is finely regulated through structural alterations of Mge1 mediated by redox levels.

Introduction

The chaperone properties of two mitochondrial proteins, Hsp70 and Ssq1p are aided by their interaction with a mitochondrial matrix protein, a “co-chaperone”, called Mge1 (Schmidt et al., 2001). This molecule is the functional homolog in Saccharomyces cerevisiae, of the bacterial co-chaperone GrpE and the human GRPEL1. There are representative homologs in archaea, eubacteria and in chloroplasts as well (Ellis, 1993; Georgopoulos and Welch, 1993; Welch, 1993). The amino acid sequence of Mge1 is given in Fig. 1A. The first 45 residues represent the mitochondrial localization sequence and they are enzymatically cleaved, inserting the remaining chain in the matrix of the organelle (Yet, the individual residues are numbered based on the entire sequence). Given that aerobic metabolism occurs in the mitochondrion, oxidative stress to its components can lead to modification in the biochemical pathways therein (Schieber and Chandel, 2014; Meng et al., 2002; Allu et al., 2015). In one of our earlier papers, we had shown how Mge1 undergoes such an oxidative modification, particularly through the oxidation of its lone methionine residue in position 155 (Met155), leading the formation of methionine sulfoxide (or sulfone), and how this process is effectively reversed by the endogenous enzyme Methionine Sulfoxide Reductase 2 (abbreviated as Mxr2), both in vivo and in vitro (Allu et al., 2015).

Studies from our laboratory and of others have shown that thermal or oxidative stress inactivates the active dimeric form of Mge1/GrpE, which slows down the Hsp70 chaperone cycle (Moro and Muga, 2006; Marada et al., 2013). Gaining insight into the structure of Mge1 is of value in understanding the underlying mechanism behind the adaptation of Mge1 to oxidative stress and its interaction with Hsp70, and Mxr2.

In this study, we investigate the effect of varying levels of oxidative stress on the conformational features of the molecule Mge1. Towards this, we have used H2O2 as the oxidant, and analyzed its effect on the molecular structure of Mge1, using biophysical and biochemical methods, molecular dynamics simulations, atomic force microscopy, and co-immuno-precipitation methods to detect the structural alterations that direct and regulate the binding of Mge1 to Mxr2 and also to Hsp70. These studies reveal that oxidized Mge1 has an open conformation with an increase in surface exposure of its otherwise compactly packed residues. Oxidation-induced weakening of the structure occurs, and selective and local structural adaptations are noticed, allowing the detachment of Mge1 from Hsp70. In addition, we show that sequestration of Mxr2 by oxidized Mge1, or vice versa, is a protective mechanism, which precludes the aggregation of Mge1, thus maintaining the chaperone cycle in order. Interestingly, oxidation of this single amino acid residue within the protein is sufficient to have pronounced effects on its intra- and inter- protein interactions, and thus its function.

Section snippets

Protein purification

Recombinant Mge1 wild type, Mge1-M155L mutant, Mxr2, and Hsp70 were purified as described earlier (Allu et al., 2015; Marada et al., 2013). Glutamate dehydrogenase (GDH) was purchased from Sigma Chemicals.

Preparation of the oxidizing agent H2O2

1M H2O2 stock solution was prepared by measuring the concentration of H2O2 spectrophotometrically using an extinction coefficient E240 = 43.6 M−1 cm−1. Native or recombinant proteins (1 mg/ml) were treated with various concentrations of H2O2 for varying time periods as described previously (

Oxidation of methionine in Mge1 disrupts its intramolecular interactions

Hydrogen peroxide in the range of 10–100 mM has been widely used to characterize the structure and function of proteins in vitro (Hayes et al., 1998; Younan et al., 2012; Allu et al., 2018). We oxidized the recombinant purified Mge1 proteins with 10 to 100 mM concentration of hydrogen peroxide. Exposing purified Mge1 to increasing levels of oxidative stress, using H2O2 as the oxidant, changes its conformation. Table 1 reveals that as H2O2 concentration increases, there is a decrease in the α-

Discussion

The 23 kDa protein Mge1 in yeast mitochondria (sequence in Fig. 1) is known to interact with the chaperone molecule Hsp70 and aid in protein folding and in the import of mitochondrial proteins. So far, Mge1 is known to be the lone “co-chaperone” for Hsp70 responding to stresses. Hence any structural perturbation to it is expected to have a deleterious effect on the chaperone function of Hsp70. Mge1 has a lone methionine residue at position 155 in its amino acid sequence. Met residues are

Acknowledgements

This work was supported by grants from the Council of Scientific and Industrial Research, India (38(1194)08/EMR-II), to N.B.V.S., and the Department of Science and Technology/Promotion of University Research and Scientific Excellence, Department of Science and Technology/Funds for Improvement of Infrastructure for Science and Technology (SR/FST/LS1-577/2016(C); (UH/DST-PURSE-2/Phase 2), and University Grants Commission/Centre of Advanced Study funds to Department of Biochemistry (

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