A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder-order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.

Tet repressor mutants with altered effector binding and allostery.

To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head-core interface on the effector specific TetR(i2) variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding. However, in TetR(i2) it leads to 4-ddma-atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures.

Structure-based design of Tet repressor to optimize a new inducer specificity.

We constructed a mutant of the tetracycline-inducible repressor protein TetR with specificity for the tc analogue 4-de(dimethylamino)anhydrotetracycline (4-ddma-atc), which is neither an antibiotic nor an inducer for the wild-type protein. The previously published relaxed specificity mutant TetR H64K S135L displays reduced induction by tc but full induction by doxycycline (dox), anhydrotetracycline (atc), and 4-de(dimethylamino)-6-demethyl-6-deoxytetracycline (cmt3). To create induction specificity for tc derivatives lacking the 4-dimethylamino grouping such as cmt3 and 4-ddma-atc, the residues at positions 82 and 138, which are located close to that moiety in the crystal structure of the TetR-[tc-Mg](+)(2) complex, were randomized. We anticipated that a residue with increased size may lead to sterical hindrance, and screening for 4-ddma-atc-specific induction indeed revealed the mutant TetR H64K S135L S138I. Out of 24 exchanges only the addition of S138I to TetR H64K S135L yielded a mutant with a pronounced reduction of affinity for atc and dox, while the one for 4-ddma-atc is not affected. The ratio of binding constants revealed a 200-fold specificity increase for 4-ddma-atc over atc. The contributions of each single mutant to specificity indicate that the tc variants bind slightly different positions in the TetR tc binding pocket.

Activity reversal of Tet repressor caused by single amino acid exchanges.

We explore by extensive mutagenesis regions in the sequence allowing reversal of the allosteric response of Tet repressor. The wild type requires anhydrotetracycline for induction. About 100 mutants are presented, which, in contrast, require the drug for repression. Their mutations are clustered at the interface of the DNA- and inducer-binding domains. This interface consists of a central hydrophobic region surrounded by several hydrogen bonds. While most of the mutants described here contain two to five mutations, we found five positions in this region of TetR, at which single amino acid exchanges lead to activity reversal. They may disrupt the hydrogen-bonding network bordering the domain interface. We assume that the mutations cause a repositioning of the DNA reading head with respect to the effector binding core so that the same conformational change can result in opposite activities.