Efficient and exclusive induction of Tet repressor by the oligopeptide Tip results from co-variation of their interaction site

Abstract

Protein-protein interactions are an important element of signal transfer within and between organisms. They are mainly mediated by short oligopeptide motifs and represent a widely used alternative to small, organic molecules for conveying information. The transcription factor TetR, a regulator of tetracycline resistance in Gram-negative bacteria, is naturally induced by tetracycline or its derivatives. The oligopeptide Tip (Transcription inducing peptide) fused to either N- or C-terminus of Thioredoxin A (TrxA) has been isolated as an artificial inducer for TetR in Escherichia coli. This inducing property can be exploited to monitor the in vivo expression of a protein of interest by fusing Tip to its C-terminus. We improve the induction efficiency of Tip by adding an aromatic amino acid before residue 1 of Tip in C-terminal fusions to TrxA. The induction efficiency of that modified TrxA-Tip fusion is further enhanced when the effector-binding pocket of TetR is enlarged by the N82A and F86A mutations. The double mutant is also insensitive to induction by tetracyclines. Thus, Tip is an exclusive inducer of this TetR variant, representing the first example of fully converting a small molecular weight effector-dependent transcription factor into one depending solely on protein-protein recognition.

Figure 1.

Induction activity of the C-terminal Met insertion mutant TrxA-Mtip. (A) A schematic presentation of the E. coli screening strain for TetR inducing peptides is shown (10). Escherichia coli DH5α(λtet50) contains lacZ under Tet control on the chromosome. Derivatives of pWH527 or pWH1413 constitutively express LacI and the TetR variants. The TetR dimer is depicted by light gray ovals with circular DNA-binding domains. TetR controls transcription of lacZ. The peptide-expressing plasmids encode either TrxA (pWH2200) or SbmC (pWH2300) or the respective fusion protein (e.g. TrxA(C)-XTip is encoded by pWH2102-X) under control of P_tac. A TetR inducing thioredoxin-peptide fusion leads to β-Gal expression as indicated by the arrow. The respective resistance genes and origins of replication of the plasmids are indicated. (B) Sequences of Tip peptides (bold) fused C-terminal or N-terminal to TrxA and the linker between TrxA and Tip are shown in one-letter abbreviations. (C) β-Gal activities obtained with the indicated TrxA-Tip fusions in the presence and absence of IPTG are shown. Gray bars show the repressed states in the absence of IPTG. Expression of the Tip-fusion proteins was induced with 60 µM IPTG (black bars). (D) Western blots indicating the steady-state levels of Tip-fusion proteins in the presence of 60 µM IPTG. Molecular weights of marker proteins (not shown) are indicated on the left side.

Figure 2.

Location of Tip in the TetR–Tip complex structure. Residues W1 to A5 of Tip and the residues N82 and F86 of TetR are displayed as gray stick models. Ac indicates the acetyl moiety at the N-terminus of Tip present in the complex. The distance (dashed lines) between the C_α of the acetyl and the C_β atoms of N82 or F86 is indicated. The dotted line indicates the continuation of Tip. The coordinates were taken from the PDB-file 2NS8 (14).

Figure 3.

Induction of TetR, TetR-N82A and TetR-F86A by C- and N-terminal TrxA-Tip fusions. The TetR variants are indicated below the bars and encoded by pWH527-derivatives. White bars show the activities induced by the C-terminal TrxA(C)-Tip fusion, gray bars those of TrxA(C)-MTip and black bars those of the N-terminal TrxA(N)-Tip fusion. The left side of the figure (labeled − IPTG) shows induction by basal expression levels while the right side (labeled + IPTG) shows the effects of induced (60 µM IPTG) expression of the Tip protein fusions.

Figure 4.

Induction of TetR and TetR-N82A by C-terminal SbmC-Tip fusions. Induction of the TetR variants indicated above the plot and encoded by pWH527-derivatives is given by the β-Gal activities expressed by the screening strain. Gray bars represent basal expression levels of SbmC(C)-Tip fusions in the absence of IPTG, while induced (60 µM IPTG) expression levels are represented by black bars. The sequence of Tip fused to SbmC is illustrated below the chart. The residues of Tip fused to SbmC are shown in bold, and the linker between SbmC and Tip is shown in light print.

Figure 5.

Induction of TetR mutants by Tip variants. Induction of TetR mutants by basal expression levels (absence of IPTG) of TrxA-XTip variants is shown. X represents any of the 20 amino acids as depicted in one-letter abbreviations (see upper right insert for the sequence). TetR variants are encoded by pWH527-derivatives. Induction of TetR variants is indicated by the corresponding β-Gal activities expressed by the screening strain. White bars show the induction values of TetR-N82A, gray bars those of TetR-F86A and black bars those of TetR-N82A-F86A. Induction by the initially analysed M insertion is highlighted by a black box. Tip indicates induction by basal expression levels of TrxA(C)-Tip and the one-letter abbreviations indicate the inserted residue. The insert in the upper left corner shows the coding of the bars. The central insert shows Western blots indicating the steady-state protein levels of TetR, TetR-N82A, TetR-F86A and TetR-N82A-F86A. The molecular mass of the TetR variants is indicated on the left.

Figure 6.

Induction of the TetR variants by tetracycline (tc), anhydro-tetracycline (atc) and doxycycline (dox). β-Gal activity was determined in the E. coli screening strain with TetR variants encoded by pWH527-derivatives. β-Gal activities are given in Miller Units (MU) with the maximal expression in the absence of TetR (w/o TetR, black bar). White bars show the expression levels obtained with the TetR variant indicated at the bottom. Light gray bars indicate the β-Gal activities after induction with 0.2 μg/ml tc, gray bars after induction with 0.2 μg/ml atc and dark gray bars after induction with 0.2 μg/ml dox.

Figure 7.

Schematic illustration of contacts made by tc and the first five residues of Tip to residues located in the TetR inducer-binding pocket. Tc (left) and the first five residues of Tip (right, one-letter abbreviations in italics) are shown as gray stick models inside the TetR inducer-binding pocket. The hydrophobic contact region of the TetR-binding pocket is shown in light gray and the two hydrophilic contact regions in gray. W1 of Tip is acetylated and the continuation of the Tip backbone is indicated as a dotted line. Mg²⁺ is shown as a gray ball and the H₂O molecules involved in Mg²⁺-coordination as black balls. Residues of TetR indicated with an apostrophe belong to the second TetR monomer in the dimer. Relevant hydrophilic interactions are indicated by dashed lines and the side chains of respective TetR amino acids are shown schematically. Residues of TetR shown solely as bold letters are positioned to make hydrophobic interactions with the inducer. Residues of TetR in normal lettering make no contact to the inducer. The PDB-files 2TRT (30) for the TetR–tc complex and 2NS8 (14) for the TetR–Tip complex were used for this presentation.