The induction of folding cooperativity by ligand binding drives the allosteric response of tetracycline repressor

Abstract

Tetracycline (Tc) repressor (TetR) undergoes an allosteric transition upon interaction with the antibiotic, Tc, that abrogates its ability to specifically bind its operator DNA. In this work, by performing equilibrium protein unfolding experiments on wild-type TetR and mutants displaying altered allosteric responses, we have delineated a model to explain TetR allostery. In the absence of Tc, we show that the DNA-binding domains of this homodimeric protein are relatively flexible and unfold independently of the Tc binding/dimerization (TBD) domains. Once Tc is bound, however, the unfolding of the DNA binding domains becomes coupled to the TBD domains, leading to a large increase in DNA-binding domain stability. Noninducible TetR mutants display considerably less interdomain folding cooperativity upon binding to Tc. We conclude that the thermodynamic coupling of the TetR domains caused by Tc binding and the resulting rigidification of the DNA-binding domains into a conformation that is incompatible with DNA binding are the fundamental factors leading to the allosteric response in TetR. This allosteric mechanism can account for properties of the whole TetR family of repressors and may explain the functioning and evolution of other allosteric systems. Our model contrasts with the prevalent view that TetR populates two distinct conformations and that Tc causes a switch between these defined conformations.

Fig. 1.

The structure of TetR. (Left) TetR consists of two monomers (light and dark blue) that interact through a four-helix-bundle. Tc (green) binds in a hydrophobic pocket in the core of each monomer. The position of residues mutated in this study, I22 (cyan), G96 (red), and L146 (orange) are displayed. (Right) A close-up of the boxed region in Left, showing an Arg residue substituted at position 96, which is the revTetR substitution. Introducing the large Arg side chain is expected to disrupt the packing of the TBD/DNA-binding domain interface residues (yellow).

Fig. 2.

Urea denaturation profiles of WT and mutant TetRs in the absence of drug. (Lower) A blow-up of the pretransition region of these profiles. This region is boxed in Upper. Protein unfolding was monitored by measuring the change in CD ellipticity at increasing concentrations of urea. The data were normalized by overlaying the main-transition region as described in Materials and Methods.

Fig. 3.

Protein unfolding of WT TetR and the ninTetR mutant in the presence of absence Tc derivatives. Urea denaturation profiles of WT (A) and ninTetR (B) in their unliganded form or in the presence of Tc or Atc were monitored by measuring CD ellipticity at increasing concentrations of urea. In B a close-up of the pretransition region is shown and the lines denoting the WT profiles from A are shown for comparison.

Fig. 4.

Folding behavior of the revTetR mutant. (A) Far UV CD scans of WT TetR and mutants in the presence or absence of Atc. (B) Urea-induced unfolding profiles for the revTetR and I22D mutants in the presence or absence of Atc.

Fig. 5.

Partial trypsin proteolysis of TetRs. The prominent digestion products are the result of cleavage in the DNA-binding domain or at the interface between the DNA-binding domain and TBD domains as determined by mass spectrometry.

Fig. 6.

In vitro operator binding by WT and mutant TetRs. (A) Electrophoretic mobility-shift assay on WT, ninTetR, I22D, and revTetR in the absence or presence of Atc. Atc was added to WT and ninTetR at a concentration of 1 μM, and assays with I22D and revTetR contained 10 μM Atc. (B) Far UV CD scans of WT and I22D TetRs in the presence or absence of tetO.

Fig. 7.

Model of ligand induced folding cooperativity in TetR. (A) The conformation of the DNA-binding domains of WT TetR in the absence of ligand is flexible as indicated by the gray blurred domains. Drug binding induces folding cooperativity between the DNA-binding and TBD domains as indicated by the black domain. Mutations (X) in the hydrophobic core of the TBD domain reduces the induced folding cooperativity between the two domains, resulting in a ninTetR. Mutations at the interface between the two domains causes destabilization of the DNA-binding domain and reduction in ligand induced folding cooperativity, creating a revTetR phenotype. Folding states that are able to bind tetO are surrounded by a red box. (B) The continuous core of TetR. The backbone of TetR is represented as a ribbon model with hydrophobic core residues (80% burial) represented in space-filling mode. The core of the DNA-binding domain is shown in yellow, the buried interface between the DNA-binding and TBD domains is shown in orange, the buried intermonomer contact region is shown in blue, and the TBD domain core is shown in red. Tc is shown in green.