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. 2008 Aug;36(13):4390-401.
doi: 10.1093/nar/gkn400. Epub 2008 Jun 28.

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

Marcus Resch  1 , Harald Striegl, Eva Maria Henssler, Madhumati Sevvana, Claudia Egerer-Sieber, Emile Schiltz, Wolfgang Hillen, Yves A Muller
Affiliations

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

Marcus Resch et al. Nucleic Acids Res. 2008 Aug.

Abstract

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.

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Figures

Figure 1.
Figure 1.
Corepression of different revTetR variants with ATC in comparison to the induction of wild-type TetR (WT TetR) analyzed using a β-galactosidase-activity reporter assay as described (16). Corepression was measured at least twice and was analyzed in vivo in media containing increasing ATC concentrations, namely 0, 0.0004, 0.001, 0.004, 0.01, 0.04, 0.1, 0.2 μM and 0.4 mM ATC. The β-galactosidase activity in the absence of TetR was set to 100% and corresponds to 8300 ± 300 units.
Figure 2.
Figure 2.
Limited proteolysis analysis of wild-type TetR and revTetR variants with subtilisin. Whereas in wild-type TetR, the presence or absence of the effector does not alter the proteolytic susceptibility of TetR, the revTetR variants are only protected against proteolysis in the presence of the effector ATC or [Mg-ATC]+. The four asterisks mark protein bands that were N-terminally sequenced.
Figure 3.
Figure 3.
Gain of secondary structure upon corepressor binding to revTetR1 monitored by CD. CD spectra of purified revTetR1 (5 µM) were recorded at 15°C before (continuous line) and after addition of ATC (broken line). Addition of ATC to revTetR1 leads to a CD spectrum that is identical to that of wild-type TetR (shown in gray). Please note that both spectra were recorded in the absence of any magnesium ions. The inset displays the difference spectrum obtained by subtracting the spectrum of ATC-bound from ATC-free revTetR1. The difference spectrum displays many characteristics of an unfolded polypeptide chain.
Figure 4.
Figure 4.
Crystal structure of the revTetR1 dimer. (A) Together with the Cα-backbone (orange), the effector molecule [Mg-ATC]+ and two additionally bound Mg2+ ions (gray) are depicted. The Cα positions of residues mutated in revTetR1, revTetR2 and revTetR4 as well as in additional TetR-variants with reverse phenotype (mutated residues 56–58) (16) are marked with black spheres. Residues at which proteolytic cleavage occurs in effector-free revTetR1 are marked in red. (B) Cartoon representation of revTetR1 colored according to the individual crystallographic thermal displacement factors (B-values) and explaining the naming of the helices in TetR (α1 to α10). In red, yellow, green and blue are displayed residues with high, medium high, medium low and low B-values, respectively. (C) The σA-weighted 2Fo−Fc electron density map around glycine 17 in revTetR1 contoured at a 1.0 σ cut-off. At the position of the missing Leu17 side-chain a water molecule binds within the interdomain interface of revTetR1.
Figure 5.
Figure 5.
Stereo representation of selected structural features in the revTetR1-[Mg-ATC]+ structure in comparison to wild-type TetR. (A) Superimposition of the region around Gly17 in revTetR1 (orange) and Leu17 in wild-type TetR (blue, PDB ID: 2TCT) (9) and tetO-bound TetR (purple, PDB ID: 1QPI) (12). Substituting Leu17 against glycine in revTetR1 does not alter the backbone conformation in any of the surrounding residues. It does, however, cause a significant reduction in the number of interside-chain interactions formed between residues from helices α1 and α4, and the α5 to α6 loop segment. Strikingly, all the residues that give rise to the reverse phenotype in the different revTetRs cluster around Leu17. (B) Comparison of the domain orientations in the revTetR1-[Mg-ATC]+ complex (orange/yellow for monomer one/two of the protein dimer), the wild-type TetR–[Mg-TC]+ complex (blue/cyan) and the tetO-bound wild-type TetR dimer (purple/pink). The structures were superimposed by matching 93 residues that immediately surround the effector-binding site. A rotation of 14.4° around axis 1 is required to orient the DNA-binding domain of revTetR1 onto that of tetO-bound wild-type TetR. Similarly, a rotation of 9.2° around axis 2 orients the DNA-binding domain of the wild-type TetR–[Mg-TC]+ complex onto that of tetO-bound wild-type TetR. The two rotation axes are clearly oriented differently but intersect in proximity of residues His63 and Ser67. This is in agreement with previous reports that indicated that in TetR these residues form a molecular hinge (12). (C) The two DNA-binding domains as observed in the revTetR1 dimer (orange/yellow) superimposed onto the corresponding domains in DNA-bound wild-type TetR (purple/pink). The superposition shows that, prior to binding, the DNA-binding domains in revTetR1 must be reoriented.
Figure 6.
Figure 6.
An orthogonal and not inverse mechanism is responsible for the effector-mediated corepression in revTetR in comparison to the induction of wild-type TetR. Whereas effector binding induces a disorder–order transition in revTetR1, which then enables effector-bound revTetR1 to interact with DNA (A–C), effector binding to wild-type TetR induces a defined conformational change in TetR that locks the protein in a conformation that is not able to interact with DNA any more (D–F). The crystal structures of revTetR1 in complex with ATC (B) and that of wild-type TetR in complex with TC (PDB ID: 2TCT) (9) (D) resemble each other. However, whereas in revTetR1 the mutations that are responsible for the reverse phenotype (black rectangles in A and B) weaken the interface between the DNA- and the effector-binding domain and thereby allow the orientation of the DNA-binding domains to freely adjust to the DNA upon binding (indicated by a double-headed arrow), in wild-type TetR the DNA-binding domains are locked in a non-DNA-binding orientation (D) because of a pendulum-like motion of helix α4 that is triggered by the binding of the effector (E–D). The structures of effector-free wild-type TetR (E) and DNA-bound effector-free wild-type TetR (F) have been sketched according to PDB-ID codes 1A6I (11) and 1QPI (12), respectively. No crystal structure is yet available for (C). The structure of (A) is inferred from CD measurements and limited proteolysis experiments. The identified cleavage sites are marked with black triangles. All DNA-binding competent structures are marked with ‘+’, those not able to bind to the operator DNA with ‘−’.
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