(Received for publication, February 9, 1996; and in revised form, March 6, 1996)
From the
Although quite common in the eukaryotic cell, bacterial proteins
with an extensive coiled-coil domain are still relatively rare. One of
the few thus far documented examples, TlpA from Salmonella
typhimurium, is characterized by a remarkably long (250 amino
acids) -helical coiled-coil domain. Herein, we demonstrate that
TlpA is a novel, sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their
corresponding protein products were purified and tested for DNA
binding. Two of the mutant proteins were shown to be deficient in DNA
binding. Both mutants were analyzed by circular dichroism and electron
microscopy, supporting the notion that mutant proteins were largely
intact despite lacking the amino acid residues necessary for DNA
binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the
repressor phenotype as a readout, the chain exchange previously
described in vitro could also be confirmed in vivo.
We believe the coiled-coil domain acts not only as a dimerization
interface but could also serve a role as a flexible modulator of the
protein-DNA interaction.
The -helical coiled-coil motif has been widely
described(1, 2) . Heptad amino acid repeats (a-b-c-d-e-f-g) are the hallmark of this structure which is
driven by apolar residues buried in a interface formed by two (or more)
-helical chains in the coiled-coil
structure(3, 4) . Positions a and d of the heptad form the characteristic 3-4 hydrophobic
repeat, which has been identified in the primary sequence of more than
200 proteins (5) .
Coiled-coils are also found as components of eukaryotic transcription factors(6) . In the eukaryotic bZip family of proteins, a coiled-coil motif of 3-4 heptads in length enables dimerization and positioning of the two polypeptide chains into a DNA binding unit(7, 8) . The involvement of the leucine zipper coiled-coil is also a centerpiece of the basic region helix-loop-helix-zipper and the basic region helix-loop-helix structures(6) . It is now evident that the coiled-coil motif is not unique to the bZip proteins, but can also be found in transcription factors with homeodomain or zinc finger DNA-binding motifs(9, 10) .
Gene regulators that utilize the coiled-coil motif appear to be less abundant in bacteria, and distinct families have yet to be recognized. To date, there are only a few documented examples of bacterial proteins per se, where the coiled-coil is a major structural feature (11, 12, 13, 14, 15) . Recently, several bacterial DNA-binding proteins with the common helix-turn-helix element have been proposed to contain a leucine zipper-like dimerization motif(16, 17, 18, 19) . Most of these bacterial examples however, lack biophysical evidence supporting the presence of a coiled-coil. Nevertheless, one cannot exclude the possibility that the leucine repeats, in these so-called zipper regions of the bacterial regulators, could mediate dimerization, if not by coiled-coil-like interaction, by way of another novel conformation. Indeed, the x-ray crystal structures of two other bacterial gene regulators, catabolite gene activator protein and the lac repressor, show that they contain short coiled-coil motifs enabling subunit interaction(20, 21) .
The TlpA protein encoded by the Salmonella typhimurium virulence plasmid forms an elongated homodimer coiled-coil(15, 22) . Here we show that TlpA has an ability to autoregulate its own gene by sequence-specific binding to its promoter DNA, an intriguing finding when one considers the sparse occurrence of extensive coiled-coils in bacterial proteins. As a first step toward dissecting the role of the coiled-coil domain in TlpA, we constructed a panel of mutant proteins lacking various portions of the reading frame. Purified mutant proteins were subjected to DNA binding and transcription assays. Based on these results we could localize the DNA-binding region, at the N terminus adjacent to the predicted coiled-coil. Evidence for in vivo chain exchange also points to TlpA's flexibility as a gene regulator.
Plasmids pMR11, p3062, and p3062d1, inclusive of tlpA or deleted fragments thereof, were available from previous work(15, 22) . In the p3062 series, tlpA is under the control of the tac promoter of pKK223-2 (Pharmacia Biotech, Inc.), whereas in the pMR series tlpA is contained in pUC19 (New England Biolabs) and expressed from its native promoter.
pMR12 and p3062d5 were manufactured by replacing in tlpA the region of codons 31 to 371 with a PCR()-generated
fragment encoding residues 43-371. The oligonucleotides used for
PCR were AGATATGGGACGAATACCAG and ACGTAAGCTTCAGGGCGTCTGAATTGTCA.
p3062d3 was produced by deleting the SalI-XhoI
fragment in tlpA of p3062. p3062d2 and p3062d4 were produced
by deleting, respectively, the 465- and 234-bp PvuII fragments
of tlpA in p3062.
The pOF tlpA-lacZ transcription fusion constructs were based on the pACYC184 vector (New England Biolabs) containing a lacZ cartridge in the BamHI-SalI sites (pKTH3090)(24) . To insert intact tlpA before lacZ tlpA was transferred as a SmaI fragment into Bluescript SK+ (Stratagene), and subsequently as a HindIII-BamHI fragment into pKTH3090 to generate pOF14. The truncated tlpA region that regulates lacZ expression in pOF6 was generated by PCR using S. typhimurium pEX102 virulence plasmid DNA as a template. Oligonucleotide CCTGGCAAGGAGAGTGGCGTGCAT was used for pOF6, and as a second primer, CAGGTCGTCGACTGTCTGCGC. Next, the resulting PCR fragment was cloned into Bluescript SK+. From the resultant plasmids the inserts were cut out as BamHI-HindIII fragments before ligation into the corresponding cloning sites of pKTH3090. Finally, all lacZ fusion constructs were supplemented with chloramphenicol acetyltransferase gene block (Pharmacia) inserted into the HindIII in front of the tlpA promoter in an opposite orientation to prevent readthrough from the plasmid.
DNase I footprinting was accomplished following manufacturers instructions utilizing the Sure Track footprinting kit (Pharmacia) with the following modification. RQ1 DNase (Promega) was used at 2 units per reaction. Each binding reaction contained about 15,000 cpm labeled fragments in gel mobility shift binding buffer at final NaCl concentration of 150 mM. The DNA used for footprinting was the 223-bp fragment produced by PCR with either one of the two oligonucleotides carrying the radioactive label. A Maxam and Gilbert G + A sequencing reaction was run for both strands. Footprinting gels were dried and either autoradiographed or analyzed by PhosphorImager (Molecular Dynamics Inc.)
Figure 1:
A, schematic representations of the
tlpA gene. The tlpA gene represented by a thin box was cloned from S. typhimurium virulence plasmid pEX102.
The SmaI fragment was cut with either SnaBI or XhoI for gel mobility shift assays to generate, respectively,
the SnaBI and the XhoI mixtures (see Fig. 2). B and C, promoterless lacZ cartridge,
represented by the large boxed symbol, was fused to suitable
sites of tlpA to generate transcriptional lacZ fusion
constructs pOF6 and pOF14. Abbreviations: ATG, initiation
codon of tlpA; P, tlpA promoter; Sma, SmaI; Sna, SnaBI; Sal, SalI; Xho, XhoI; lacZ, -galactosidase promoterless
gene
Figure 2: Gel mobility shift assay with the tlpA gene fragments. Fragments derived from differentially digested tlpA SmaI block, with either XhoI or SnaBI, were radiolabeled and mixed with TlpA or only the binding buffer. TlpA addition leads to a disappearance of the 5` end fragment in tlpA. Abbreviations: X, no protein added; T, TlpA; Xh, XhoI; Sn, SnaBI
Figure 3: A, schematic representation of deleted regions in TlpA amino acid sequence. Internal (dTlp1-dTlp5) deletion derivatives of TlpA are shown underneath with the line symbol indicating residues present in each protein. All deletions are in frame, i.e. the proteins are translated in their entirety but lacking the residues indicated. B, plot of the probability of coiled-coil formation. Probability P(S) is shown as a function of amino acid residue number in TlpA protein. Figure was calculated and produced with the COILS2 program(5, 11) .
Figure 4: Gel mobility shift assays. A, specificity controls for TlpA binding. TlpA was mixed with either the putative target or noncognate DNA and subjected to a mobility shift assay. Same DNA fragments were used at more than 600-fold excess to compete the binding. B, binding of TlpA mutant proteins to target DNA competed with noncognate DNA. Abbreviations: T, TlpA; T1, dTlp1; T2, dTlp2; T3, dTlp3; T4, dTlp4; C, control DNA fragment; F, DNA fragment which includes the target.
Figure 5: DNase I footprinting of the tlpA 5` region. A, a 223-bp cognate DNA fragment was labeled differentially to probe both strands (lanes 1-4 and 5-8). Maxam-Gilbert G + A reaction was run to enable sequence recognition (lanes 1 and 5). Abbreviations: X, no protein added; T, TlpA; T1, dTlp1; G, G + A sequencing reaction B, nucleotide sequence of the tlpA 5` end is shown where the region identified in protection assays is marked by a line above or below the corresponding strand. In the sequence the transcription start site is marked by a +1(22) , and the arrow marks the first translated codon (ATG) in tlpA.
Figure 6: Gel mobility shift assay showing the loss of function in dTlp1 and dTlp5. Abbreviations: X, no protein added; T, TlpA; T1, dTlp1; T5, dTlp5
Figure 7: Circular dichroism spectra recorded of wild-type TlpA (open circles) and the deletion mutants, dTlp5 (open triangles) and dTlp1 (open squares). Spectra were recorded from 186 to 260 nm at 0.5-nm increments at 25 °C in 50 mM phosphate buffer pH 7.0, 150 mM NaCl. Inset shows an SDS-polyacrylamide gel electrophoresis of purified proteins used throughout these studies.
Figure 8: Electron microscopy of TlpA and the two nonbinding mutants showing protein-protein interaction leading to a formation of filaments and filament networks. A, TlpA; B, dTlp1; C, dTlp5
Previously we have described that S. typhimurium virulence plasmid codes for a coiled-coil protein TlpA which is a temperature-dependent chain-exchanging entity as shown in our in vitro system and is also able to form oligomeric structures resembling intermediate filaments in morphology(15, 22) . The biological function (if any) of oligomerization remains at present unresolved, yet our new finding that TlpA is a gene regulator fits well with the flexibility offered by the monomer exchange phenomenon.
The sequence specificity of binding
by TlpA is clearly demonstrated by footprinting and gel mobility shift
assays. Binding was directed to specific target DNA, and could be
competed only with a fragment containing this sequence (Fig. 4A). The inability to see a well resolved
TlpADNA complex in gel mobility shift assays could indicate that
protein-protein associations produce different sized oligomers (15) which dissipate the label throughout the running lane. The
most apparent explanation for this is that a dimer binds DNA and
undergoes higher oligomer interactions, producing complexes with
different compositions. Then again, in vivo, the amount of
TlpA may be so low that the issue of oligomerization may be redundant.
Footprinting also showed a preferred region of interaction (Fig. 5) and identified the -10 and -35 elements in
the broad protected region. Hydroxyl radical footprinting will
hopefully shed more light on whether the large footprint is due to
steric hindrance caused by TlpA or binding to several operator sites in
the promoter region.
With our mutant-protein panel we have demonstrated the localization of the DNA-binding region with respect to the coiled-coil domain and also begun to probe the role of the latter in the binding. The only residues whose deletion leads to abolished DNA binding are those that map to the N-terminal portion of TlpA, adjacent to the predicted coiled-coil as delineated from lack of binding by dTlp1 and dTlp5 (Fig. 6). Deletions within the coiled-coil most adjacent to the DNA-binding region produced a different complex in the gel mobility shift assay as shown by dTlp2 and dTlp3 (Fig. 4). This can be interpreted as either a higher oligomer stabilization at the expense of any smaller complexes, or more likely as some loss of binding specificity, i.e. more protein is bound per DNA, suggesting coiled-coil serves a role in positioning the binding regions. Such an assumption is supported by the fact that dTlp4, with a considerable deletion in the coiled-coil but more distant to residues implicated in binding, showed a gel mobility shift pattern which was similar in appearance to that produced by the wild-type TlpA (Fig. 4).
The capacity for oligomerization can be ruled out as a single key element of importance for binding since the DNA binding-deficient forms showed also a tendency for organized higher order protein-protein interactions. Based on the dominant negative phenotype of dTlp5 (and dTlp1, data not shown) we believe the binding structure to be minimally a dimer. From previous studies we can conclude that dTlp1 (referred to previously as the 41-kDa protein) (15) is able to form parallel unstaggered dimers with TlpA. dTlp5, which has a smaller deletion at the N terminus, shows a CD spectra nearly identical to TlpA and dTlp1, therefore we believe it also readily forms dimers (dTlp5 is rapidly oxidized into disulfide bridge dimers; data not shown). Also, the oligomer assembly of dTlp5 shows the capacity for formation of filament stacks in electron microscopy. It is unlikely, yet possible, that the inability to see a fully developed filament network in dTlp5 reflects an effect of the deleted residues, because dTlp1 with an overlapping but larger deleted segment shows all forms of oligomer arrangement. In any case, electron microscopy shows clearly that the fibrous appearance is intact, and this would not be expected in a randomly folded polypeptide preparation (nor would the highly helical CD spectra). Residues deleted in dTlp5 serve as a road map for identification of all of the critical residues which are needed for specific DNA interaction. We do not know to what extent the 13 amino acids are representative of the residues critical to function in the N-terminal DNA-binding region and whether the DNA-binding residues form an independent domain or are an extension of the coiled-coil. More detailed studies are underway to reveal the nature of the DNA-binding domain in TlpA, which could represent a novel combination of a DNA-binding structure coupled to a coiled-coil. At present we are unable to find any significant homology to known DNA-binding motifs in any part of tlpA sequence. Collectively our data indicates that TlpA consists of a functional outline where the N terminus is responsible for DNA binding and the adjacent long coiled-coil serves to dimerize the binding interfaces and position them for sequence specific contacts.
Transcription assays have shown that the region bound by TlpA contains an active promoter which can be repressed by TlpA in trans, but not by dTlp5 (Table 1). These findings also set the stage for testing for in vivo chain exchange. Previously, we had shown that TlpA dimers at 37 °C are capable in monomer exchange with related partners, underscoring the dynamic nature of this protein(15) . The transcription reporter construct pOF14 which is little active by itself, when propagated in a cell which also produces the DNA-binding mutant dTlp5, is activated to levels exhibited by the nonrepressed tlpA promoter. This can be easily explained if one considers that for every TlpA translated from the pOF14 transcript there is bound to be a dTlp5 partner protein for dimer formation or chain exchange between homodimers. Such heterodimers would be composed of one wild-type monomer with an intact recognition half-site and one monomer lacking this region. This is analogous to eukaryotic CHOP or Id proteins which have a defective and a nonexistent DNA-binding domain, respectively, and form inactive heterodimers with other partners and thereby block transcription factors from binding to their targets(28, 29) . The heterodimerization in the case of TlpA points also to the importance of the coiled-coil in organizing the structure into a binding proficient form.
A gene regulator such as TlpA would certainly have a role in the pathogenesis of virulent bacteria such as S. typhimurium, which is under constant pressure to sense its environment before entering the host, and while in the host as it progresses from one niche to another along its route of invasion experiencing changes in pH, temperature, and osmolarity(30) . Coiled-coil structures which are known from many studies to respond to changes in the environment(31) , could be ideal sensors to variations in the intracellular environment. An elevated temperature or osmolarity could affect the interactions within the coiled-coil domain and be sensed directly by the cytoplasmic TlpA. Temperature can of course influence TlpA activity, since there likely exists a dimer-monomer equilibrium in the cell, and by raising the temperature the equilibrium could be shifted more toward the monomer which could not bind DNA by itself. Ongoing studies are aimed at addressing the question of inducing signals and the targets for TlpA interaction. Finally, it is tempting to speculate that bacteria encode a set of gene regulators that exploit the coiled-coil motif and heterodimerization capacity. Another candidate for this new protein family is the E. coli protein, KfrA, also characterized by an extensive coiled-coil domain and an ability to autoregulate itself (14) .