Molecular Effects of Elongation Factor Ts and Trigger Factor on the Unfolding and Aggregation of Elongation Factor Tu Induced by the Prokaryotic Molecular Chaperone Hsp33.

Minho Keum,Sung-Hee Lee,Kyeong-Hyeon Yoon,Young-Ho Lee,Wookyung Yu,Hyung-Sik Won,Jihoon Kim,Yuxi Lin,Mi-Seong Kim,Dai Ito,Ji-Hun Kim

doi:10.3390/biology10111171

Abstract

Simple SummaryProteins are versatile biological macromolecules involved in most biological processes. However, because of the highly labile nature of protein structures, protein quality control (PQC) to ensure proteostasis (i.e., protein homeostasis)is difficult. Therefore, proteins of a specialized class (i.e., molecular chaperones) are required that assist in proper folding and prevent aberrant folding of other proteins. Hsp33 was originally discovered as a holding chaperone that is overexpressed upon heat shock and activated by oxidation to prevent the misfolding of client proteins. Recently, an unfoldase/aggregase activity of Hsp33 was identified in its reduced state against a specific substrate, EF-Tu, which plays a key role in protein biosynthesis in cells. The present study demonstrates that EF-Tu unfolding/aggregation by Hsp33 can be accelerated by another molecular chaperone trigger factor. Given that the unfolded/aggregated EF-Tu is finally degraded by another chaperone, Lon protease, it is likely that a chaperone network dysregulating EF-Tu operates in heat shock to attenuate protein biosynthesis, which is harmful to cell survival under stressed conditions. Therefore, the apparently contradictory chaperone function (i.e., promotion of client misfolding) of Hsp33 can also be associated with the PQC processes to ensure proteostasis in cells.Hsp33, a prokaryotic redox-regulated holding chaperone, has been recently identified to be able to exhibit an unfoldase and aggregase activity against elongation factor Tu (EF-Tu) in its reduced state. In this study, we investigated the effect of elongation factor Ts (EF-Ts) and trigger factor (TF) on Hsp33-mediated EF-Tu unfolding and aggregation using gel filtration, light scattering, circular dichroism, and isothermal titration calorimetry. We found that EF-Tu unfolding and subsequent aggregation induced by Hsp33 were evident even in its complex state with EF-Ts, which enhanced EF-Tu stability. In addition, although TF alone had no substantial effect on the stability of EF-Tu, it markedly amplified the Hsp33-mediated EF-Tu unfolding and aggregation. Collectively, the present results constitute the first example of synergistic unfoldase/aggregase activity of molecular chaperones and suggest that the stability of EF-Tu is modulated by a sophisticated network of molecular chaperones to regulate protein biosynthesis in cells under stress conditions.

Highlights

As protein structures are labile in nature and aberrant protein folding is highly deleterious to cells, protein quality control (PQC) to ensure cellular proteostasis is critical for cell viability [1,2,3]
The molecular interactions of elongation factor Tu (EF-Tu), elongation factor Ts (EF-Ts), heat shock protein 33 (Hsp33), and trigger factor (TF) were investigated via gel filtration assay to monitor EF-Tu oligomerization (Figure 1)
To examine the effect of EF-Ts on the Hsp33-mediated EF-Tu oligomerization, we confirmed that EF-Tu and EF-Ts formed a stable one-to-one complex (Figure 1b), as is generally known [27], whereas no significant interaction was observed between EF-Ts and Hsp33 (Figure 1c)

Summary

Introduction

As protein structures are labile in nature and aberrant protein folding is highly deleterious to cells, protein quality control (PQC) to ensure cellular proteostasis (i.e., protein homeostasis) is critical for cell viability [1,2,3]. The conserved cysteines in the zinc-binding redox-switch domain form disulfide bonds with concomitant release of zinc, which results in the unfolding of the C-terminal redox-switch domain and the middle linker domain [12,13,14,15,16]. This partially-unfolded Hsp functions as a holding chaperone that binds to unfolding intermediates of client proteins to prevent further progression of misfolding and promote native folding [17,18,19]. Dimer and highorder oligomer formation of activated Hsp enhances its chaperone activity [16,20,21]

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Results

Conclusion