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

Klotzsche M, Goeke D, Berens C, Hillen W - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.

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.

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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.
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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.

Mentions: There is a clear distinction among the hydrophobic amino acids between aromatic and aliphatic residues. C-terminal TrxA(C)-XTip variants with V, L or I show a decrease in activity compared to M, while variants with H, F, Y or W induce TetR more efficiently. Comparison of the contacts in the TetR inducer-binding pocket to tc and the first five residues of Tip (Figure 7) shows that most of the key interactions for tc (H64, N82, F86, H100, P105) are not formed with Tip (14). In particular, the space occupied by the A-ring of tc appears to be empty in the Tip–TetR complex. It is thus conceivable that the aromatic residues mimic the A-ring structure of tc best, followed by the flexible M residue with the easily polarizable sulphur, while the large hydrophobic residues fit least well.Figure 7.


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

Klotzsche M, Goeke D, Berens C, Hillen W - Nucleic Acids Res. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1919500&req=5

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.
Mentions: There is a clear distinction among the hydrophobic amino acids between aromatic and aliphatic residues. C-terminal TrxA(C)-XTip variants with V, L or I show a decrease in activity compared to M, while variants with H, F, Y or W induce TetR more efficiently. Comparison of the contacts in the TetR inducer-binding pocket to tc and the first five residues of Tip (Figure 7) shows that most of the key interactions for tc (H64, N82, F86, H100, P105) are not formed with Tip (14). In particular, the space occupied by the A-ring of tc appears to be empty in the Tip–TetR complex. It is thus conceivable that the aromatic residues mimic the A-ring structure of tc best, followed by the flexible M residue with the easily polarizable sulphur, while the large hydrophobic residues fit least well.Figure 7.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Institut für Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.

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.

Show MeSH
Related in: MedlinePlus