Combinations of the α-Helix−Turn−α-Helix Motif of TetR with Respective Residues from LacI or 434Cro:  DNA Recognition, Inducer Binding, and Urea-Dependent Denaturation

Abstract

We constructed 10 different variants of TetR by substituting all or some of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro. The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro. The urea-induced denaturation of the 10 variants occurs in single reversible transitions, which are centered around 4.3 M urea. Denaturation is concentration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers. An analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typical for dimeric proteins of this size. Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol. These results suggest that the DNA reading head of Tet repressor is a thermodynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dimerization domains of the individual monomers. Variants containing replacements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever. We attribute this to the alteration of the two N-terminal residues in this alpha-helix. TetR variants were active in nonspecific DNA binding, when either all or only the solvent-exposed residues in the recognition alpha-helix of HTH were exchanged to the respective LacI sequence. Replacement of the same residues by the respective amino acids from 434Cro yielded hybrid proteins that specifically recognize tetO in vitro. Taken together, these results establish that the similarity of operator recognition between 434Cro and TetR is greater than between TetR and LacI and confirm that prediction of the recognized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix.

Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113

“…First, the sequence and structure of the phl operator, phO, are very similar to those of the tetO operator located within the tetR-tetA intergenic region (2). Second, the putative helix-turn-helix motif present at the amino terminus of the PhlF protein (4,12,54) shows interesting similarities with the helix-turn-helix of TetR.…”

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

“…2, WT TetR displayed a major unfolding transition between 4 and 6 M urea. This transition has been shown to be a reversible two-state reaction that primarily reflects the concerted and reversible unfolding of the TBD domain from a folded dimer to two unfolded monomers (23)(24)(25). Although a TetR mutant completely lacking its DNA-binding domain (⌬DNB mutant) showed a major unfolding transition at a similar position as WT TetR, striking differences were observed in the shape of the unfolding curves of WT TetR and the ⌬DNB mutant at urea concentrations Ͻ4 M, which we refer to as the pretransition region (Fig.…”

Solvent-exposed Residues in the Tet repressor (TetR) Four-helix Bundle Contribute to Subunit Recognition and Dimer Stability

“…Renaturation was achieved by incubating the samples overnight at 8 M urea and then diluting them 200-fold with F-buffer. Thermodynamic calculations were done as described before (18).…”

“…The latter mediates tetracycline binding and dimerization via a four-helix bundle formed by the helices ␣8 and ␣10 of each subunit. Disruption of the dimeric structure of TetR by urea concomitantly leads to denaturation of the subunits (18).…”

“…2), and the midpoint of the unfolding transition depends on the protein concentration (data not shown), as expected for a bimolecular reaction. Thus, as for TetR(B) (18), unfolding of the TetR(B/D) chimera is quantitatively described by a two-state model, in which only folded dimers and unfolded monomers exist at equilibrium in significant concentrations. Calculated thermodynamic stabilities of the TetR(B/D) variants are also given in Table II responsible for the increased stability of TetR(B/D)128,179-184, but the nature of that interaction cannot be concluded from the current data.…”