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Journal of Bacteriology, December 2008, p. 8238-8243, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00889-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Altered Oligomerization Properties of N316 Mutants of Escherichia coli TyrR

Takashi Koyanagi,^1,2 Takane Katayama,² Hideyuki Suzuki,^1,3 and Hidehiko Kumagai^2*

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502,¹ Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, 921-8836,² Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan³

Received 30 June 2008/ Accepted 6 October 2008

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The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrR^E274Q, a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrR^N316D and TyrR^N316R mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrR^N316D and TyrR^N316R, respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrR^N316D.

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TyrR is a transcriptional regulator of genes mainly involved in the metabolism of aromatic amino acids in bacteria. Of the known TyrR proteins, Escherichia coli TyrR is the most extensively studied and is known to regulate expression of at least eight genes responsible for biosynthesis and transport of aromatic amino acids (1, 4, 5, 11, 12, 16, 18, 19, 22, 23, 25-29) (Fig. 1A). In addition, it has recently been shown that the folA gene, encoding dihydrofolate reductase, is also regulated by TyrR (29). The protein comprises three domains with different functions (Fig. 1B). The N-terminal domain has a site for aromatic amino acid binding (23) and a region that interacts with the -subunit of RNA polymerase to stimulate transcription initiation (12). The TyrR homolog of Haemophilus influenzae lacks this domain, and therefore it is incapable of transcriptional activation (9, 10, 24, 30, 31). The central domain shows a high sequence similarity to those of ⁵⁴-dependent enhancer-binding proteins (NtrC family), but TyrR differs from them in that it regulates transcription from ⁷⁰-dependent promoters to reflect the lack of the GAFTGA motif that is essential for contact with the ⁵⁴ subunit. The NtrC family, including TyrR, belongs to the AAA+ (ATPases associated with diverse cellular activities) superfamily (4, 15, 22). A common feature of the AAA+ superfamily is the formation of a ring-shaped oligomer in response to environmental stimuli (15). TyrR exists as a dimer in solution, but in the presence of ATP and tyrosine (or a high concentration of phenylalanine) it changes its conformation from a dimer to a hexamer (1, 11, 25, 26). This central domain-dependent oligomerization is triggered by the binding of aromatic amino acids to another binding site located in this domain (an ATP-dependent site) (25). The C-terminal domain has a helix-turn-helix motif, which is structurally similar to that of the cyclic AMP receptor protein (3), and binds to DNA with a consensus sequence of TGTAAAN₆TTTACA (TyrR box) (Fig. 1A) (19).

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FIG. 1. (A) The promoter-operator regions of the TyrR regulon. TyrR binding sites are represented by black (strong boxes) and white (weak boxes) rectangles. Transcription initiation sites (+1) and the –35 and –10 promoter regions are indicated. Regulatory modes of TyrR on the respective promoters in the presence of tyrosine (Tyr) and phenylalanine (Phe) are indicated as R (repression) or A (activation). (B) Domain structure of TyrRE. coli. The numbering starts at the initiation codon. Roles of the respective domains are indicated. The ATP-binding Walker A and B motifs and the helix-turn-helix DNA-binding motif (HTH) are shown by black and shaded boxes, respectively, and the glutamic acid-274 (E274) and asparagine-316 (N316) residues are indicated. aroF, tyrosine-repressible 3-deoxyarabinoheptulosonate 7-phosphate synthase; aroG, phenylalanine-repressible 3-deoxyarabinoheptulosonate 7-phosphate synthase; aroL, shikimate kinase II; aroP, aromatic amino acid permease; folA, dihydrofolate reductase; mtr, tryptophan-specific permease; tyrB, aromatic amino acid aminotransferase; tyrP, tyrosine-specific permease; tyrR, transcriptional regulator TyrR of E. coli K-12 MG1655; tpl, tyrosine phenol lyase of E. herbicola.

The promoter-operator regions of the TyrR regulon encompass one to three TyrR boxes with different affinities, as summarized by Pittard et al. (18) (Fig. 1A). A box to which a TyrR dimer can bind in the absence of an aromatic amino acid coeffector usually has high sequence identity with the consensus and is called a strong box. On the other hand, a site having low sequence identity is called a weak box. TyrR binds to the weak box only when a strong box is juxtaposed nearby on the same face of the helix and when the TyrR dimer bound to the strong box forms an oligomer in the presence of coeffectors (cooperative binding). TyrR regulates transcription either positively or negatively by changing its dimer-hexamer conformation and by binding to the strong/weak boxes (Fig. 1A).

In a previous study, Kwok et al. found that the substitution of glutamine for glutamic acid at position 274 (E274Q) of E. coli TyrR renders the protein deficient in oligomer formation (11). This mutant TyrR exhibited normal binding to ATP, but the tyrosine-mediated hexamer formation was severely impaired, which suggested an important role for this residue in the process of dimer-hexamer conversion. In the present study, we tried to isolate second-site suppressors of this mutant protein and identified asparagine-316 as a critical residue in the fine-tuning of the oligomeric state of TyrR.

Mutations that suppress the inability of Erwinia herbicola TyrR^E275Q to activate the tpl promoter. We have previously studied the TyrR protein of Erwinia herbicola (6, 8, 21). E274 of E. coli TyrR corresponds to E275 of E. herbicola, and the corresponding mutant (TyrR^E275Q) showed an impaired ability to activate tpl (Table 1), possibly due to a deficiency in hexamer formation (the action mechanism of TyrR on the tpl promoter is described later), and was targeted for isolation of a second-site suppressor by introducing random mutations.

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TABLE 1. Amino acid substitutions that suppress the impaired ability of E. herbicola TyrRE275Q to activate tpl

A mutant tyrR^E275Q_{E. herbicola} library was screened for a recovered ability of its product to activate the (tpl'-'lac) gene (pTK871) (6). Error-prone PCR and subsequent construction of the plasmid library was performed as described previously (6) except that pTK815 (p15A replicon bla⁺ tyrR^E275Q_{E. herbicola}) was used as the template for PCR. The plasmid library was used to transform E. coli tyrR strain TK596 [F^– ara (lac-pro) thi tyrR::kan⁺ (srl-recA)306::Tn10] (6) carrying pTK871. Transformants were visually screened for enhanced formation of blue color on LB plates (14) containing 1 mM 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and four transformants were selected for further analysis. Sequence analysis of the four mutant tyrR genes revealed that all alleles still contained the E275Q substitution; nevertheless, the abilities of their products to activate tpl were recovered to a level comparable to that of wild-type TyrR_{E. herbicola} (Table 1). The most increased expression was observed for the strain with tyrR9, which carried an A503T substitution in the DNA-binding domain. In the solution structure of the DNA-binding domain of H. influenzae TyrR, the corresponding residue (A306) lies in the C-terminal -helix (PDB accession code 1G2H) and could be buried in the major groove of the double-stranded DNA. Therefore, it is likely that the A503T substitution affects the DNA-binding property of TyrR, but the reason why this amino acid replacement rescued the ability of TyrR^E275Q_{E. herbicola} to activate tpl is unclear. The other three tyrR alleles carried a common mutation leading to an amino acid substitution of aspartic acid for asparagine-324 (N324D). Since N324 is located in the AAA+ domain and is conserved in all TyrR proteins identified so far, we hypothesized that this residue might be involved in the oligomerization process. Therefore, subsequent studies were focused on this residue.

Regulatory properties of the mutant TyrR proteins in vivo. First, we constructed various N324 mutants of E. herbicola TyrR to examine their in vivo regulatory properties; however, the E. coli strain expressing TyrR^N324D_{E. herbicola} was unable to grow in the minimal medium without the addition of aromatic amino acids or shikimic acid, an intermediate of aromatic amino acid biosynthesis (data not shown), suggesting that the mutant protein severely repressed the genes responsible for aromatic amino acid biosynthesis (17). We decided to use E. coli TyrR instead of E. herbicola TyrR for further analysis because the corresponding E. coli TyrR mutant (TyrR^N316D) did not cause such severe growth retardation. Site-directed mutagenesis of the tyrR_{E. coli} gene was carried out by the QuikChange method (Stratagene) using pTK723 (p15A replicon bla⁺ tyrR_{E. coli}) (6) as a template and oligonucleotides with the desired mutations. The entire fragment used for later manipulation was sequenced to ensure that no base changes other than those planned had occurred.

Three genes (aroF, tyrP, and tpl) of the TyrR regulon were translationally fused to lac genes (6) and used as reporters. The aroF and tyrP genes of E. coli encode tyrosine-repressible 3-deoxyarabinoheptulosonate 7-phosphate synthase and tyrosine-specific permease, respectively (17-19), and the tpl gene of E. herbicola encodes tyrosine phenol lyase (6-8). In the cases of aroF and tyrP a strong box(es) is located upstream of, and a weak box overlaps with, the –35 promoter (Fig. 1A). When the ligand-mediated cooperative binding of TyrR occurs in this region, RNA polymerase is eliminated from the promoter, which results in repression of expression (26). In the presence of tyrosine, TyrR represses the expression of aroF and tyrP, while in the presence of phenylalanine TyrR represses aroF but activates tyrP (26, 28). Phenylalanine-mediated activation of tyrP occurs when the ligand binds to the N-terminal ATP-independent site of the TyrR dimer bound to the strong box (23). Whereas hexamer formation of TyrR causes repression of aroF and tyrP, the tpl promoter is activated by ligand-mediated hexamerization of TyrR bound to the three distant boxes (Fig. 1A) (2, 8, 20). Activation of tpl also requires the binding of the aromatic amino acid to the N-terminal ATP-independent site.

A plasmid carrying one of three reporter genes [(aroF'-'lac) (pTK588), (tyrP'-'lac) (pTK589), or (tpl'-'lac) (pTK871)] (6) was introduced into the E. coli strains with the mutant tyrR genes on a compatible plasmid (Fig. 2 legend). These strains were grown in M63-0.2% (wt/vol) glucose minimal medium (14) supplemented with 1 µg/ml thiamine-HCl and 30 µg/ml proline in the absence and presence of 1 mM phenylalanine or tyrosine. The amount of each TyrR^N316 variant in the cells was essentially the same, as revealed by immunoblotting using an anti-TyrR antibody (data not shown).

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FIG. 2. Mode of regulation by the wild-type and mutant TyrR proteins. pTK479 (p15A replicon bla+) (none) (6), pTK723 (p15A replicon bla+ tyrR+E. coli) (wild type) (6), or pTK723-derived plasmids with the mutant tyrRE. coli genes were introduced into strain TK809 [F– ara (lac-pro) thi (srl-recA)306::Tn10 tyrR::cat+] carrying (aroF'-'lac) (A) or (tyrP'-'lac) (B). pYG541 (pSC101 replicon bla+) (none), pYG543 (pSC101 replicon bla+ tyrR+E. coli) (wild type), or pYG543-derived plasmids with the mutant tyrRE. coli genes were introduced into strain TK596 (see text) carrying (tpl'-'lac) (C). Cells were grown in the absence (open bars) or presence of 1 mM phenylalanine (filled bars) or tyrosine (shaded bars), and β-galactosidase activities were measured. Assays were carried out in duplicate for at least two separate cultures, and results shown are means ± standard deviations.

N316 substitutions had varied effects on the regulatory properties of TyrR, and among the mutants, significant changes were observed for TyrR with N316D/E and N316R/K substitutions (Fig. 2). The basal expression levels of (aroF'-'lac) and (tyrP'-'lac) in the cells carrying TyrR^N316D_{E. coli} were significantly lower than those in the cells carrying wild-type TyrR (Fig. 2A and B). In the presence of phenylalanine, TyrR^N316D_{E. coli} slightly activated aroF expression, unlike TyrR^WT_{E. coli}, and stimulated tyrP transcription, similar to TyrR^WT_{E. coli}. In both cases, the expression levels were considerably lower than those for the cells carrying wild-type TyrR (Fig. 2A and B). The ratio of phenylalanine-mediated activation of tpl by TyrR^N316D_{E. coli} was 2.5-fold higher than that caused by TyrR^WT_{E. coli} (Fig. 2C). The aroF and tyrP expression levels in the cells carrying TyrR^N316D_{E. coli} dropped to a basal level in the presence of tyrosine, and the expression levels were almost equal to those observed for the cells carrying wild-type TyrR grown in the presence of tyrosine. Activation of tpl in the presence of tyrosine was enhanced in the cells carrying TyrR^N316D_{E. coli} compared with the cells carrying wild-type TyrR. These results, i.e., the significant decrease of aroF and tyrP expression levels and increase in tpl expression, suggested an enhanced ability of this mutant TyrR to self-associate. In any case, the N316D substitution overcame the effects caused by the E274Q substitution (E274Q versus N316D E274Q), indicating that N316D is a second-site suppressor mutation of E274Q. In the strain carrying TyrR^N316E_{E. coli}, a similar regulatory mode was observed for aroF, tyrP, and tpl expression levels (Fig. 2A, B, and C). It seems that replacement of N316 with acidic residues promotes self-association of the TyrR protein.

By contrast, the basal expression levels of aroF and tyrP were derepressed in the strains carrying TyrR^N316K_{E. coli} or TyrR^N316R_{E. coli} compared with the strains with wild-type TyrR, and the expression levels were almost equal to those of the tyrR-null mutants (Fig. 2A and B). In the cells carrying TyrR^N316K/R_{E. coli}, no repressive effect was observed for aroF expression in the presence of phenylalanine, but significant repression was observed in the presence of tyrosine (Fig. 2A). The ratio of activation of tyrP in the presence of phenylalanine was enhanced in the cells carrying N316K/R mutants compared with that in the cells with wild-type TyrR, but the ratio of repression in the presence of tyrosine was slightly decreased (Fig. 2B). The basal expression of tpl did not change between the cells carrying wild-type and N316K/R mutant TyrR proteins, but the ligand-mediated activation was considerably lower in the cells carrying TyrR^N316K/R_{E. coli} compared with the cells carrying TyrR^WT_{E. coli} (Fig. 2C). These results, even though tyrosine-mediated repression of aroF and tyrP was still observable, suggested a diminished ability of the mutant TyrR proteins to form an oligomer.

TyrR^N316L also derepressed aroF and tyrP expression levels to a lesser extent but did not affect tpl expression (Fig. 2). Replacement of N316 with alanine, cysteine, or histidine did not significantly alter the regulatory property of the E. coli TyrR protein (Fig. 2).

Oligomerization of E. coli TyrR^WT, TyrR^N316D, and TyrR^N316R proteins. The TyrR^N316D_{E. coli} and TyrR^N316R_{E. coli} proteins showed significantly altered in vivo regulatory properties; therefore, we analyzed their oligomer formation abilities in the presence of the ligands using gel filtration chromatography, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 3). To obtain the purified proteins, the respective tyrR genes were placed under the control of a T7 promoter (pET-3a; Novagen), and the resulting plasmids were introduced into a DE3-lysogenized tyrR-deficient derivative of BL21 [F^– hsdS(r_B^– m_B^–) gal ompT tyrR::kan⁺ (DE3)] (YG110). The purification process was essentially the same as that described by Argaet et al. (1) except that Superdex 200 HR10/30 (GE Healthcare) was used instead of Superose 12.

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FIG. 3. Gel filtration analysis of the wild-type TyrRE. coli (A), TyrRN316DE. coli (B), and TyrRN316RE. coli (C) proteins. Fifteen µl of each fraction (0.3 ml, total fraction volume) was subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, followed by Coomassie brilliant blue R-250 staining. Ferritin (molecular weight [MW], 440,000) and aldolase (MW, 160,000) were used as molecular weight markers (GE Healthcare). The prestained protein marker Broad Range (New England BioLabs) was electrophoresed next to the samples as shown at the left and center of the gel (M; bands for MW 62,000 and 47,500 are visible).

Size exclusion chromatography (Superdex 200 HR10/30 column) was carried out by injecting a protein solution (1 ml) containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM EDTA, 0.1 mM dithiothreitol, 25 µg/ml phenylmethylsulfonyl fluoride, 10 mM MgCl₂, and purified TyrR (5 µM) with or without 100 µM ATP (or ADP) and the indicated concentrations of tyrosine (Tyr) or phenylalanine (Phe). Elution was done at a flow rate of 0.25 ml/min in the same buffer without phenylmethylsulfonyl fluoride at 4°C. None of the TyrR proteins oligomerized without the addition of nucleotides, even in the presence of aromatic amino acids (Fig. 3). With respect to wild-type TyrR, oligomer formation was observed in the presence of both ATP and aromatic amino acids Phe or Tyr (Fig. 3A). However, in the case of TyrR^N316D_{E. coli}, oligomerization was observed in the presence of ATP alone. The presence of aromatic amino acids apparently enhanced the stable hexamer formation of the TyrR^N316D_{E. coli} protein compared with wild-type TyrR (Fig. 3A versus B). These results are in agreement with the altered regulatory function of TyrR^N316D_{E. coli} seen in vivo, i.e., the enhanced ability of the mutant protein to form a hexamer led to the repression of aroF and tyrP expression and activation of tpl. To our surprise, TyrR^N316D_{E. coli} shifted its size to a higher molecular weight in the presence of ADP and Phe or Tyr, whereas wild-type TyrR absolutely requires ATP for its oligomerization (4) (Fig. 3A versus B). Even in the presence of ADP alone, a slight shift in size was observed for TyrR^N316D_{E. coli}, suggesting that ADP could serve, if only partially, as an alternative to ATP for this mutant protein in the oligomerization process. The conformational change of this mutant protein in the presence of aromatic amino acids was reversible (data not shown).

In contrast, TyrR^N316R_{E. coli} was eluted at a position corresponding to a dimer under all conditions tested, though a slight shift in size was seen when the tyrosine concentration was raised to 750 µM (Fig. 3C). These properties also explain the altered regulatory mode of this protein in vivo, i.e., derepression of aroF and tyrP and decreased activation of tpl.

Concluding remarks. N316 is located just downstream of the Walker B motif in the central domain of E. coli TyrR and is conserved in all the TyrR proteins isolated so far. In this study, we demonstrated that the N316 substitution had varied effects on the in vivo regulatory properties of TyrR, and we also showed the altered in vitro oligomerization properties of TyrR^N316D and TyrR^N316R. Although we have not examined the DNA-binding properties of the N316 variants and thus could not attribute the altered in vivo regulatory modes only to their anomalous oligomerization properties, it is established that the N316 residue is crucial for the protein to fine-tune its oligomeric state in response to the ligands. It should be noted that the amino acid residues at this position vary among the other NtrC family members (A in E. coli NtrC, Q in Salmonella enterica serovar Typhimurium ZraR and Klebsiella pneumoniae NifA, and E in Sinorhizobium meliloti DctD, E. coli PspF, and Aquifex aeolicus NtrC1) and that the corresponding residue of A. aeolicus NtrC1 (E256) forms a hydrogen bond with the sensor II residue from a neighbor subunit (PDB code 1NY6) (13). The sensor II residue is thought to be critical for discrimination of ATP/ADP-bound forms. Considering the oligomer formation of TyrR^N316D in the presence of ADP and Phe or Tyr, it is interesting to speculate that the role of N316 is as a sensor II modulator, although the interaction of N316 and the sensor II residue (R417) of E. coli TyrR has not been established. Further in vitro studies are required to elucidate the role of the residue in the oligomer formation process of TyrR.

 
This work was supported by a Grant-in-Aid for Scientific Research by Young Scientists [(B)18780060] from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. T. Koyanagi was supported by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science and Technology.

 
* Corresponding author. Mailing address: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan. Phone: 81-76-227-7522. Fax: 81-76-227-7557. E-mail: hidekuma{at}ishikawa-pu.ac.jp

Published ahead of print on 17 October 2008.

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Journal of Bacteriology, December 2008, p. 8238-8243, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00889-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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