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

SIMPLE SUMMARY: Proteins 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. ABSTRACT: 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.