Conversion of the allosteric transition of GroEL from concerted to sequential by the single mutation Asp-155 -> Ala

Abstract

The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 --> Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 --> Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes.

Fig. 1.

Ribbon diagram of the structure of two adjacent GroEL subunits (PDB code 1OEL) (33) showing the location of the inter-subunit Arg-197–Glu-386 salt bridge and the intra-subunit Asp-155–Arg-395 salt-bridge. The equatorial, intermediate, and apical domains of the two subunits are shown in different shades of green, gold, and blue, respectively. Helices G and M are shown as red and yellow cylinders, respectively. Single-letter notation for amino acids is used.

Fig. 2.

Initial velocities of ATP hydrolysis by the Phe-44 → Trp single mutant (A) and the Phe-44 → Trp, Asp-155 → Ala double mutant (B) as a function of ATP concentration. The reactions were carried out as described in Materials and Methods. The data for the Phe-44 → Trp single mutant (taken from ref. 22) and the Phe-44 → Trp, Asp-155 → Ala double mutant were fitted to a Hill-type equation for two allosteric transitions (24) or Eq. 1 for three allosteric transitions, respectively. (Insets) The data for low ATP concentrations.

Fig. 3.

Observed rate constants of the ATP binding-induced T to R conformational change of the Phe-44 → Trp single mutant and the Phe-44 → Trp, Asp-155 → Ala double mutant as a function of ATP concentration. The reactions were carried out as described in Materials and Methods. The data for the Phe-44 → Trp single mutant (A) and Phe-44 → Trp, Asp-155 → Ala double mutant (B) were fitted to a Hill-type equation for two allosteric transitions (22) or Eq. 2 for three allosteric transitions, respectively.

Fig. 4.

Representative class averages of single-ring GroEL particles containing the Phe-44 → Trp single mutation or the Phe-44 → Trp, Asp-155 → Ala double mutation in the presence of different concentrations of ATP. The number of particles corresponding to each class average is indicated. The respective class averages for the Phe-44 → Trp single mutant and the Phe-44 → Trp, Asp-155 → Ala double mutant, in the absence of ATP, are shown in A and B. The respective class averages for the Phe-44 → Trp single mutant and the Phe-44 → Trp, Asp-155 → Ala double mutant, in the presence of 100 μM ATP, are shown in C and D. Class averages for the Phe-44 → Trp mutant, in the presence of 5 μM ATP, are shown in E. The class averages for the Phe-44 → Trp, Asp-155 → Ala mutant in the presence of 5 μM ATP are shown in F. Image processing was carried out as described in Materials and Methods.

Fig. 5.

Extent of 7-fold symmetry of class averages of single-ring GroEL particles containing the Phe-44 → Trp single mutation or the Phe-44 → Trp, Asp-155 → Ala double mutation in the presence of different concentrations of ATP. Each class average was rotated by N (an integer that runs from 1 to 360) × 1°. The extent of the 7-fold symmetry of the different class averages was determined by calculating a cross-correlation coefficient, r, between the nonrotated class average and the rotated ones by using procedures written with spider (27). The respective rotational correlation plots for the Phe-44 → Trp single mutant and the Phe-44 → Trp, Asp-155 → Ala double mutant, in the absence of ATP, are shown in A and B. The respective rotational correlation plots for Phe-44 → Trp single mutant and the Phe-44 → Trp, Asp-155 → Ala double mutant, in the presence of 100 μM ATP, are shown in C and D. The respective rotational correlation plots for the Phe-44 → Trp single mutant and the Phe-44 → Trp, Asp-155 → Ala double mutant, in the presence of 5 μM ATP, are shown in E and F.