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

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.

Keywords: EF-Ts; EF-Tu; Hsp33; aggregase activity; molecular chaperone; protein biosynthesis; proteostasis; trigger factor; unfoldase activity.

Figure 1

Oligomerization of EF-Tu detected via gel filtration assay. Gel-filtration (HiLoad 16/600 Superdex 200 column at 1 mL/min of flow rate) profiles are shown for EF-Tu (green in a,b,e,g), Hsp33 (blue in a,c,f), EF-Ts (gray in b,c), TF (gray in e,f), EF-Tu:Hsp33 (red in a,d,g), EF-Tu:EF-Ts (black in b,d), EF-Tu:TF (black in e), Hsp33:TF (orange in f), EF-Tu:EF-Ts:Hsp33 (purple in d), and EF-Tu:TF:Hsp33 (purple in g). Underneath the gel-filtration profiles, SDS-PAGE images are shown for individual fractions of gel filtration (lane R, mixture of the individual proteins before incubation; M, molecular size marker in kDa): EF-Tu (a) Hsp33 (a) EF-Ts (b) TF (e) EF-Tu:Hsp33 (a) EF-Tu:EF-Ts (b) EF-Tu:TF (e) EF-Ts:Hsp33 (c) TF:Hsp33 (f) EF-Tu:EF-Ts:Hsp33 (d) and EF-Tu:TF:Hsp33 (g). Sample solutions for the gel filtration contained 0.1 mM of each protein, which was pre-incubated at 35 °C for 30 min before loading (1 mL of injection volume) onto the column.

Figure 2

Aggregation of EF-Tu detected via light scattering. Light scattering intensity (arbitrary unit, a.u.) at 400 nm was recorded for 10 min, including the pre-incubation (2 min) and reaction monitoring (8 min) periods. Time-course change of the light scattering intensity (ΔLS) is shown for indicated protein samples. EF-Tu (green, red, purple, and orange) and EF-Tu:EF-Ts complex (blue) were incubated at 35 °C, followed by equimolar (15 μM) titration (designated as the time 0 min) of Hsp33 (red and blue), TF (orange), or both (purple). Control experiments for EF-Tu alone (green) and Hsp33 alone (light blue in the inset) were performed by adding blank buffer, whereas TF alone is presented in the pre-incubation period for subsequent addition of Hsp33 (pink in the inset).

Figure 3

Conformational change of EF-Tu detected via CD spectroscopy. Far-UV CD spectra (main panel) were measured for the protein samples indicated in parentheses before and after 30-min incubation at 35 °C. The CD intensities measured for diluted samples (10, 5, and 3 μM for EF-Tu, EF-Tu:Hsp33, and EF-Tu:Hsp33:TF, respectively) before and after the incubation, which was performed with 0.2 mM of each protein, were normalized as molar ellipticity ([θ]). The inset shows time-dependent change of molar ellipticity (Δ[θ]) at 222 nm, for the indicated protein samples (15 μM) during incubation at 35 °C in the CD cell.

Figure 4

Calorimetric characterization of the TF binding to EF-Tu. (a) ITC thermograms obtained by a single-shot titration (added at 5 min of incubation at 33 °C) of Hsp33 (upper) and equimolar Hsp33:TF mixture (lower) to EF-Tu are shown. Differential power (i.e., heat flow) is denoted as DP. The earlier (negative) and later (positive) ITC peaks were indicated by ΔH_front and ΔH_back, respectively. (b) ITC thermograms (upper) obtained at 20 °C by titrating TF to native EF-Tu (left) and ^OligoEF-Tu (right) are shown with binding isotherms (lower), where the black line indicates a fitting curve and the inset table presents the thermodynamic parameters determined for the binding of TF to ^OligoEF-Tu.