Concerted ATP-induced allosteric transitions in GroEL facilitate release of protein substrate domains in an all-or-none manner

Abstract

The double-ring chaperonin GroEL mediates protein folding, in conjunction with its helper protein GroES, by undergoing ATP-induced conformational changes that are concerted within each heptameric ring. Here we have examined whether the concerted nature of these transitions is responsible for protein substrate release in an all-or-none manner. Two chimeric substrates were designed, each with two different reporter activities that were recovered after denaturation in GroES-dependent and independent fashions, respectively. The refolding of the chimeras was monitored in the presence of GroEL variants that undergo ATP-induced intraring conformational changes that are either sequential (F44W/D155A) or concerted (F44W). Our results show that release of a protein substrate from GroEL in a domain-by-domain fashion is favored when the intraring allosteric transitions of GroEL are sequential and not concerted.

Fig. 1.

Models of the EGFP–rhodanese and mDHFR–rhodanese chimeras. (A) In the EGFP–rhodanese chimera, the C terminus of EGFP (green) is connected to the N terminus of rhodanese (red) by a linker peptide of 11 residues (white). The N terminus of EGFP is fused to a peptide of 39 aa with an N-terminal His₆-tag (yellow). (B) In the mDHFR–rhodanese chimera, the C terminus of mDHFR (cyan) is joined to the N terminus of rhodanese (red) via a linker peptide (white) of 15 residues that contains His₆-tag. Rhodanese (29), EGFP (19), and mDHFR (30) are represented by ribbon diagrams of their crystal structures (Protein Data Bank codes 1RHD, 1EMA, and 1U70, respectively). In the case of mDHFR, the only available crystal structure corresponds to a mutant ternary complex. The N and C termini of the chimeras are indicated.

Fig. 2.

Reactivation of the denatured EGFP–rhodanese and mDHFR–rhodanese chimeras under different conditions. (A) Yields of reactivation of the EGFP–rhodanese chimera in the absence of GroE (spontaneous) and in the presence of GroEL (F44W or F44W/D155A) and 1 mM ATP with or without GroES. The extent of reactivation was determined 4 h after initiation of folding by measuring rhodanese activity (red bars) and EGFP fluorescence (green bars). Each experiment was repeated four times, and standard deviations were determined. The final concentration of the chimera was 0.5 μM. (B) Yields of reactivation of the mDHFR–rhodanese chimera in the absence of GroE (spontaneous) and in the presence of GroEL (F44W or F44W/D155A) and 1 mM ATP with or without GroES. The extent of reactivation was determined 3 h after initiation of folding by measuring rhodanese (red bars) and mDHFR (blue bars) activities. Each experiment was repeated four times, and standard deviations were determined. The final concentration of the chimera was 0.5 μM. (C) Time-resolved regain of the EGFP fluorescence of the chimera in the presence of the F44W and F44W/D155A GroEL mutants and different concentrations of ATP. Denatured chimera and the F44W (filled symbols) or F44W/D155A (open symbols) GroEL mutants were mixed and incubated for 7 min before addition of 50 μM (circles) or 1 mM (squares) ATP. The addition of 50 μM ATP was followed by a second addition of 950 μM ATP to these samples at the time indicated by the arrow. (D) Relative yields of the EGFP fluorescence of the chimera in the presence of the F44W or F44W/D155A GroEL mutants at different concentrations of ATP. The denatured chimera and the F44W (filled circles) or F44W/D155A (open circles) GroEL mutants were mixed, and refolding was then initiated by addition of ATP and allowed to proceed until maximum reactivation was reached. The yields of refolded EGFP were normalized relative to the maximum yield that was obtained at 1 mM ATP and corrected for spontaneous refolding, which is reflected in the slow phase in C and is seen before addition of ATP. The data for the F44W and F44W/D155A mutants were fitted to Hill equations for one or two (31) allosteric transitions, respectively (the data for the F44W/D155A mutant cannot be fitted well to the Hill equation for one allosteric transition). (Inset) A magnification of the plot at low ATP concentrations. (E) Relative yields of refolding of mDHFR in the chimera in the presence of the F44W (filled squares) or F44W/D155A (open squares) GroEL mutants at different concentrations of ATP. The yields were normalized, and the data were fitted as described in the legend to D. Spontaneous refolding before addition of ATP as in C was not observed. (F) Relative yields of refolded EGFP (circles) and mDHFR (squares) in the presence of the F44W (filled symbols) or F44W/D155A (open symbols) GroEL mutants at different concentrations of ATP. Denatured EGFP or mDHFR (not fused to rhodanese) and the F44W or F44W/D155A GroEL mutants were mixed, and refolding was then initiated by addition of ATP and allowed to proceed until maximum reactivation was reached. The yields of refolded substrate are normalized relative to the maximum yields obtained at high ATP concentrations. The data were fitted to the Hill equation for one allosteric transition. Note that maximum yields are reached at ATP concentrations that are lower than those at which maximum yields of the chimeras are reached (D and E).

Fig. 3.

Gel filtration of the products of GroEL-assisted reactivation of the denatured EGFP–rhodanese chimera. (A and B) Elution profiles of the products of the chimera refolding reaction in the presence of 1 mM ATP and the respective GroEL mutants F44W or F44W/D155A. (C) The column was calibrated by separating the native chimera that was mixed with 1 μM F44W GroEL and 0.2 mM ATP in G10K buffer. The peaks from left to right correspond to GroEL, the chimera, and ATP, respectively. The elution profiles were determined by measuring the absorbance at 280 nm (black line) and the fluorescence of EGFP (green circles).

Fig. 4.

Scheme showing coupling between ATP-induced conformational changes in GroEL and release and folding of EGFP in the EGFP–rhodanese chimera. The ATP-induced conformational change of a subunit from the t state (with low affinity for ATP and high affinity for nonfolded substrates) to the r state (with high affinity for ATP and low affinity for nonfolded substrates) is represented by a counterclockwise rotation that causes the protein substrate-binding site (black) to face away from the cavity. The wild-type variant (F44W) of GroEL and the F44W/D155A mutant undergo ATP-induced concerted (t₇→r₇) and sequential (e.g., t₇→t₄r₃→r₇) allosteric transitions, respectively. The color-coding of the unfolded and folded (ribbon diagram of EGFP) parts of the chimera are according to Fig. 1A. For simplicity, only the substrate-bound ring of GroEL is shown in this scheme.