Intricate Interactions within the ccd Plasmid Addiction System
2002; Elsevier BV; Volume: 277; Issue: 5 Linguagem: Inglês
10.1074/jbc.m105505200
ISSN1083-351X
AutoresMinh‐Hoa Dao‐Thi, Daniël Charlier, Remy Loris, Dominique Maes, Joris Messens, Lode Wyns, Jan Backmann,
Tópico(s)Bacteriophages and microbial interactions
ResumoThe ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA, and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA·CcdB·DNA complex with a CcdA:CcdB ratio of one. This (CcdA2CcdB2)n complex has multiple DNA-binding sites and spirals around the 120-bp promoter region. The ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA, and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA·CcdB·DNA complex with a CcdA:CcdB ratio of one. This (CcdA2CcdB2)n complex has multiple DNA-binding sites and spirals around the 120-bp promoter region. high performance liquid chromatography isothermal titration calorimetry reverse phase chromatography 3-(N-morpholino)propanesulfonic acid N,N-bis(2-hydroxyethyl)glycine The 95-kb low copy number F plasmid is maintained inEscherichia coli with remarkable stability. Many synergistic processes are responsible for its maintenance in the bacterial population. The plasmid contains a partitioning system to distribute plasmid copies to the daughter cells during cell division as well as several site-specific recombination systems to resolve oligomeric plasmid molecules. In addition, the F plasmid and other low copy number plasmids encode programmed cell death systems: daughter cells, which did not inherit the plasmid, are killed. Such systems are called post-segregational killing or addiction systems (reviewed in Ref. 1Engelberg-Kulka H. Glaser G. Annu. Rev. Microbiol. 1999; 53: 43-70Crossref PubMed Scopus (311) Google Scholar). The F plasmid encodes three such systems: srn (stable RNA degradation) (1Engelberg-Kulka H. Glaser G. Annu. Rev. Microbiol. 1999; 53: 43-70Crossref PubMed Scopus (311) Google Scholar, 2Nielsen A.K. Thorsted P. Thisted T. Wagner E.G. Gerdes K. Mol. Microbiol. 1991; 5: 1961-1973Crossref PubMed Scopus (44) Google Scholar), flm (F leading maintenance) (1Engelberg-Kulka H. Glaser G. Annu. Rev. Microbiol. 1999; 53: 43-70Crossref PubMed Scopus (311) Google Scholar,3Golub E.I. Panzer H.A. Mol. Gen. Genet. 1988; 214: 353-357Crossref PubMed Scopus (25) Google Scholar), and ccd (controlled cell death) (1Engelberg-Kulka H. Glaser G. Annu. Rev. Microbiol. 1999; 53: 43-70Crossref PubMed Scopus (311) Google Scholar, 4Miki T. Chang Z.T. Horiuchi T. J. Mol. Biol. 1984; 174: 627-646Crossref PubMed Scopus (56) Google Scholar, 5Miki T. Yoshioka K. Horiuchi T. J. Mol. Biol. 1984; 174: 605-625Crossref PubMed Scopus (78) Google Scholar). The ccd system was the first one to be identified (6Hagihara Y. Oobatake M. Goto Y. Protein Sci. 1994; 3: 1418-1429Crossref PubMed Scopus (44) Google Scholar) and remains the best studied. The ccd operon encodes a toxin (CcdB: 101 amino acids, 11.7 kDa) and its antidote (CcdA: 72 amino acids, 8.3 kDa). The synthesis of the ccd proteins is autoregulated at the level of transcription by a complex of both toxin and antitoxin (7de Feyter R. Wallace C. Lane D. Mol. Gen. Genet. 1989; 218: 481-486Crossref PubMed Scopus (50) Google Scholar, 8Tam J.E. Kline B.C. Mol. Gen. Genet. 1989; 219: 26-32Crossref PubMed Scopus (70) Google Scholar, 9Tam J.E. Kline B.C. J. Bacteriol. 1989; 171: 2353-2360Crossref PubMed Google Scholar). Both proteins are expressed, the toxic activity of CcdB being reversibly inactivated by the presence of CcdA. The stability (10Dao-Thi M.H. Messens J. Wyns L. Backmann J. J. Mol. Biol. 2000; 299: 1393-1406Crossref Scopus (29) Google Scholar) as well as in vivo life span (11Jaffé A. Ogura T. Hiraga S. J. Bacteriol. 1985; 163: 841-849Crossref PubMed Google Scholar) of CcdB is higher than that of CcdA. It was postulated by us that the thermodynamic stability of CcdA is low enough to keep the protein close to unfolding in vivo conditions, whereby it facilitates its metabolization (10Dao-Thi M.H. Messens J. Wyns L. Backmann J. J. Mol. Biol. 2000; 299: 1393-1406Crossref Scopus (29) Google Scholar). Upon plasmid loss, CcdA is quickly degraded by the Lon protease (12Van Melderen L. Bernard P. Couturier M. Mol. Microbiol. 1994; 11: 1151-1157Crossref PubMed Scopus (195) Google Scholar, 13Van Melderen L. Dao Thi M.-H. Lecchi P. Gottesman S. Couturier M. Maurizi M.R. J. Biol. Chem. 1996; 271: 27730-27738Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), leaving CcdB free to kill the cell. CcdB acts as a poison and inhibitor of DNA gyrase, an essential enzyme that catalyzes negative supercoiling of DNA (14Bernard P. Couturier M. J. Mol. Biol. 1992; 226: 735-745Crossref PubMed Scopus (366) Google Scholar, 15Kampranis S.C. Howells A.J. Maxwell A. J. Mol. Biol. 1999; 293: 733-744Crossref PubMed Scopus (38) Google Scholar, 16Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar). CcdA inhibits the lethal action of CcdB by directly binding the toxin (inactivation) and by the extraction of the toxin from its complex with the target gyrase (rejuvenation) (17Maki S. Takiguchi S. Horiuchi T. Sekimizu K. Miki T. J. Mol. Biol. 1996; 256: 473-482Crossref PubMed Scopus (40) Google Scholar, 18Bernard P. Kezdy K.E. Van Melderen L. Steyaert J. Wyns L. Pato M.L. Higgins P.N. Couturier M. J. Mol. Biol. 1993; 234: 534-541Crossref PubMed Scopus (155) Google Scholar). The crystal structures of CcdB as well as that of a gyrase fragment have been solved and a model for the CcdB-gyrase complex proposed (19Loris R. Dao-Thi M.H. Bahassi E.M. Van Melderen L. Poortmans F. Liddington R. Couturier M. Wyns L. J. Mol. Biol. 1999; 285: 1667-1677Crossref PubMed Scopus (124) Google Scholar, 20Critchlow S.E. O'Dea M.H. Howells A.J. Couturier M. Gellert M. Maxwell A. J. Mol. Biol. 1997; 273: 826-839Crossref PubMed Scopus (69) Google Scholar, 21MoraisCabral J.H. Jackson A.P. Smith C.V. Shikotra N. Maxwell A. Liddington R.C. Nature. 1997; 388: 903-906Crossref PubMed Scopus (399) Google Scholar). Still, crucial mechanistic aspects of the ccd system have remained unrevealed. Even some basic parameters such as the stoichiometry and binding constants of the intermolecular interactions involved are unknown. In the present paper, we investigate in detail the interactions between CcdA, CcdB, and specific operator DNA using a range of biophysical and biochemical techniques. The purification of CcdA and CcdB was carried out as described before in (19Loris R. Dao-Thi M.H. Bahassi E.M. Van Melderen L. Poortmans F. Liddington R. Couturier M. Wyns L. J. Mol. Biol. 1999; 285: 1667-1677Crossref PubMed Scopus (124) Google Scholar). Electrospray mass spectrometry was carried out in a Quattro II quadrupole mass spectrometer (Micromass, Manchester, UK) having a m/z range of 4000, equipped with an electrospray interface as described previously (22Messens J. Hayburn G. Desmyter A. Laus G. Wyns L. Biochemistry. 1999; 38: 16857-16865Crossref PubMed Scopus (51) Google Scholar). The binding between CcdA and CcdB was monitored in near-UV CD in the spectral range from 280 to 295 nm. The protein solutions were centrifuged and filtered (0.45 μm) to remove turbidity. Approximately 4 ml (exact volume determined using analytical balances) of ∼10 μm CcdB (concentration determined photometrically using the extinction coefficients from Dao-Thi et al. (10Dao-Thi M.H. Messens J. Wyns L. Backmann J. J. Mol. Biol. 2000; 299: 1393-1406Crossref Scopus (29) Google Scholar)) in the corresponding buffer (either 50 mm citrate, pH 5.6, 100 mm NaCl, or 50 mm cacodylate, pH 6.5, 100 mm NaCl) was placed in a thermostated cuvette with 1-cm optical path length and then titrated with ∼60 μl of ∼35 μm CcdA (exact concentration determined photometrically prior to titration) solution in the same buffer so that a molar ratio CcdB:CcdA of 2 was reached after around 10 additions. The progress of reaction was best monitored by the intensity of the negative peak at around 283 nm. HPLC1chromatography was carried out on a 600S Controller coupled to a 996 PDA detector (Waters, Milford, MA) equipped with a Rheodyne 9125 (Cotati, CA) injector using a reverse phase C4 column (4.6 × 25 mm) (214TP54) (Vydac, Hesperia, CA) equilibrated in 15% acetonitrile, 0.1% trifluoroacetic acid at 1 ml/min. The column was developed with a 50-min linear gradient from 10 to 50% acetonitrile at room temperature. Absorption data collection at 280 nm was performed under Millennium (Waters). The column was calibrated with 1:1 and 1:2 mixtures of CcdA-CcdB. Peak heights were found to be the most accurate to calculate CcdA:CcdB ratios and were used as such. For the size-exclusion experiments the CcdA:CcdB 1:2 complex was prepared by adding dropwise CcdA to CcdB in different buffer solutions to achieve a final concentration of 5 and 10 μm,respectively. The 500-μl mixture was incubated for 10 min at room temperature prior to injection (450 μl) on a Superdex75 HR 10/30 size-exclusion column (Amersham Biosciences, Inc., Uppsala, Sweden). The buffer solutions are: 50 mm sodium citrate, pH 5.0, 50 mm sodium cacodylate, pH 6.0, 50 mmMops, pH 7.0, 50 mm Tris, pH 8.0, and 50 mmBicine, pH 9.0. The Superdex75 HR column was, respectively, equilibrated in 50 mm buffer solution, 150 mmKCl, 0.1 mm EDTA, and calibrated with a gel filtration standard from Bio-Rad, i.e. γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12(1.35 kDa). All runs were performed at room temperature on anÄkta-Explorer (Amersham Biosciences, Inc.). For the DNA complex binding experiments: the Superdex75 HR column was equilibrated with 50 mm Tris, pH 8.0, 150 mm KCl, 0.1 mm EDTA. The CcdA:CcdB 1:1 complex was prepared by adding dropwise CcdA to CcdB in 50 mm Tris, pH 8.0, to achieve a final concentration of 15 μm each. The CcdA·CcdB·DNA complex was prepared by adding dropwise CcdA to the mixture of CcdB and the 85-bp MH12 DNA fragment (see Fig.5C) in 50 mm Tris, pH 8.0, to achieve a final concentration of 15 μm CcdA, 15 μm CcdB, and 80 μg/ml DNA or 5 μm CcdA, 10 μmCcdB, and 80 μg/ml DNA. In another experiment the CcdA2CcdB4 complex was formed, followed by a 10-min incubation with 60 μg/ml 85-bp MH12 DNA fragment in 50 mm Tris, pH 8.0. Each mixture (500 μl) was incubated for 10 min at room temperature prior to injection (450 μl). The procedure of preparing the mixtures is critical and has to be followed accurately to avoid possible precipitation. Binding of CcdA and CcdB proteins to specific 5′-end-labeled DNA fragments was determined according to the method described by (23Fried M.G. Crothers D.M. Nucleic Acids Res. 1983; 11: 141-158Crossref PubMed Scopus (101) Google Scholar) with modifications. Protein-DNA complexes were formed in 20 μl of binding buffer (10 mm Tris-HCl, pH 7.4, 250 mm KCl, 5 mm MgCl2, 2.5 mm CaCl2, 0.5 mm dithiothreitol, 2.5% glycerol) in the presence of 0.1 μg of sonicated herring sperm DNA for 20 min at 37 °C. Samples were loaded on preelectrophoresed 6% (w/v) polyacrylamide gels in TBE buffer (89 mm Tris, 89 mm boric acid, 2.5 mm EDTA). Electrophoresis was performed in the same TBE running buffer at room temperature at 8 V/cm for 3 h. DNase I footprinting was performed according to the method described by Galas and Schmitz (24Galas D.J. Schmitz A. Nucleic Acids Res. 1978; 5: 3157-3170Crossref PubMed Scopus (1330) Google Scholar). Purified proteins were incubated with 5′-end-labeled DNA fragments in 100 μl of binding buffer (see above, gel retardation) and further treated as described previously (25Charlier D. Hassanzadeh G. Kholti A. Gigot D. Pierard A. Glansdorff N. J. Mol. Biol. 1995; 250: 392-406Crossref PubMed Scopus (68) Google Scholar). Binding studies with CcdA and CcdB were carried out with a MicroCalTM Omega isothermal titration calorimeter. The concentration of the samples in the cell and the syringe was determined spectrophotometrically. Both proteins were dialyzed prior to titration against the same buffer using Spectra/Por® CE (molecular weight cut-off, 5000; sample volume, 2 ml) at room temperature (cold room temperature causes precipitation of CcdA at high concentration) for 3 h. The titrations were carried out at a temperature of 25 °C. Due to the complexity of the interaction, the standard software could not be applied meaningful. The results were interpreted based on the inflection point and the shape of the thermograms. Semiquantitative conclusions on the strength of the involved microscopic interactions were obtained supposing that in the early stage of a titration, one type of interaction dominates the overall reaction. Differential scanning calorimetry thermograms were simulated for different values of p =K·Ctot·n, whereK is the binding constant, Ctot the macromolecular concentration in the cell before starting the titration, and n is the number of binding sites. Only for pvalues of around 5–50 sigmoidal curves are obtained. For values around unity and below, the titration curve is featureless. For pvalues of 100 or above, an abrupt transition ("box car") is observed. From the shape of the experimentally measured thermograms, the parameter p is about 2000 for the forward titration (CcdA into CcdB) and 20 for the reverse titration (CcdB into CcdA). This leads to the following limits for the binding constants involved: 105m−1 < KL< KH < 108m−1. The following model, where B2 represents the CcdB dimer and A2 the CcdA dimer, was set up. A2+B2↔KLA2B2A2B2+A2↔KLA2B2A2A2B2+B2↔KHB2A2B2A2B2+A2B2↔KHA2B2A2B2A2B2A2+B2↔KHA2B2A2B2B2A2B2+A2↔KLA2B2A2B2B2A2B2+A2B2↔KHB2A2B2A2B2A2B2A2+A2B2↔KHA2B2A2B2A2A2B2A2B2+B2↔KHB2A2B2A2B2A2B2A2B2+A2↔KLA2B2A2B2A2⋯etc.MODEL1Higher aggregates thanA2B2A2B2A2andB2A2B2A2B2were neglected. These aggregates could already be considered to have the tendency to precipitate. Combining the above expressions for the equilibrium constants and the following mass balances.A2,total=[A2]+KL[A2][B2]+KLKH[A2][B2]2+2KL2[A2]2[B2]+2KHKL2[A2]2[B2]2+3KHKL3[A2]3[B2]2+2KH2KL2[A2]2[B2]3(…)Equation 1 B2,total=[B2]+KL[A2][B2]+KL2[A2]2[B2]+2KLKH[A2][B2]2+2KHKL2[A2]2[B2]2+2KHKL3[A2]3[B2]2+3KH2KL2[A2]2[B2]3(…)Equation 2 The equilibrium concentrations were solved numerically using the Euler method. Iterations were carried out to minimize the differences between the calculated A2,total andB2,total and the actual values. The robustness of the model was checked by applying different binding constants within the same order of magnitude, giving essentially the same results. Two series of simulations were carried out. In the first one the total concentration of CcdB (B2,total) was kept constant at 10−5m dimer equivalents (0.47 mg/ml), and the total CcdA concentration (A2,total) was varied from the thousandth to the thousandfold. In a second series of simulation the CcdA concentration was restrained and the amount of CcdB altered in an analogous way. CcdA is thought to counteract the lethal effect of CcdB by forming a noncovalent complex that prevents CcdB to interact with gyrase (17Maki S. Takiguchi S. Horiuchi T. Sekimizu K. Miki T. J. Mol. Biol. 1996; 256: 473-482Crossref PubMed Scopus (40) Google Scholar,18Bernard P. Kezdy K.E. Van Melderen L. Steyaert J. Wyns L. Pato M.L. Higgins P.N. Couturier M. J. Mol. Biol. 1993; 234: 534-541Crossref PubMed Scopus (155) Google Scholar). The stoichiometry of this complex is still not strictly defined (8Tam J.E. Kline B.C. Mol. Gen. Genet. 1989; 219: 26-32Crossref PubMed Scopus (70) Google Scholar, 17Maki S. Takiguchi S. Horiuchi T. Sekimizu K. Miki T. J. Mol. Biol. 1996; 256: 473-482Crossref PubMed Scopus (40) Google Scholar, 26Van Melderen L. Analysis of the regulatory role of Lon protease in the activation of the ccd system of programmed cell death. Université Libre de Bruxelles, Brussels, Belgium1995Google Scholar), and both CcdA2CcdB2 and CcdA2CcdB4 have been suggested (16Maki S. Takiguchi S. Miki T. Horiuchi T. J. Biol. Chem. 1992; 267: 12244-12251Abstract Full Text PDF PubMed Google Scholar, 28Bahassi E.M. Salmon M.A. Van Melderen L. Bernard P. Couturier M. Mol. Microbiol. 1995; 15: 1031-1037Crossref PubMed Scopus (52) Google Scholar). We have therefore used high resolution gel filtration chromatography experiments to observe such complexes and to determine experimental conditions suitable for a detailed characterization of their properties. CcdA:CcdB 1:2 and 1:1 mixtures were prepared and analyzed on an analytical gel-permeation column Superdex75 HR at different pH values (pH 5 to pH 9). For the 1:2 mixture, three major populations were observed (Fig.1): a 65.5-kDa peak (at an elution volume of 10 ml) that is in agreement with a CcdA2CcdB4 complex, a 24.4-kDa (elution volume 12 ml) peak corresponding to the dimer of CcdB, and a peak around 18-ml elution volume that contained aggregated and degraded CcdA and CcdB. The 16.6-kDa peak of the dimer of CcdA was only observed at pH 5. The 65.5-kDa peak was analyzed on a C4-reverse phase column (C4-RPC), and its chromatographic profile at 280 nm is shown on theinset in Fig. 1. The peaks obtained from the reverse phase step were analyzed by electrospray mass spectrometry. CcdA elutes in two peaks with retention times of 39.4′ and 39.8′, both with identical masses of 8372 Da, while CcdB elutes with a retention time of 48.6′ with a mass of 11704 Da. The masses determined with electrospray mass spectrometry match the calculated masses of CcdA and CcdB. After calibration of the C4-RPC column for CcdA and CcdB, the ratio of CcdA:CcdB under the 65.5-kDa peak was found to be 1:2, confirming a CcdA2CcdB4 complex. The height of the gel filtration peak at around 18-ml elution volume is pH-dependent. It is largest in the experiment carried out at pH 9 (Fig. 1). Reverse phase chromatography on a C4-RPC showed that this peak also contains a mixture of intact CcdA and CcdB. The ratio of CcdA:CcdB under this elution peak is 2:3, which might correspond to a CcdA4CcdB6 complex. The fact that this peak is eluting before the salt peak of the size-exclusion column means that this aggregate is aspecifically interacting with the matrix or is able to enter the pores of the matrix. When CcdA and CcdB were mixed in a 1:1 molar ratio, a completely different elution profile was obtained. The chromatogram of this mixture at pH 8.0 is also shown in Fig. 1. A broad peak around an elution volume of 12 ml was observed, indicating the presence of different possible complexes together with free CcdB and CcdA. A peak around an elution volume of 18 ml containing aggregates was also observed. CcdA has a flat and featureless CD spectrum in the region from 260 to 300 nm, allowing the titration of CcdA into CcdB to be followed in the near-UV CD spectrum of CcdB. As can be seen in Fig.2A, two peaks between 280 and 300 nm shift in intensity as well as position (from 288.9 to 292.2 nm and from 282.2 to 283.8 nm). Fig. 2B shows the change in intensity of these two peaks when titrating CcdA into CcdB. A plateau is reached around a CcdA:CcdB ratio of 2. Around this ratio, addition of CcdA also causes local clouding. At a CcdA:CcdB ratio of 3 a persistent turbidity evolves. This coincides with a sagging of the intensity of the CD spectra. Similar, when adding CcdB to a CcdA solution, turbidity evolves at a very early stage. Furthermore isothermal titration calorimetry (ITC) was used to study the interaction between CcdA and CcdB. ITC has the advantage that it does not depend on the optical properties of a system but records a physical property inherent to almost all binding processes, the production or absorption of heat. Hence titrations can be carried out in both directions, i.e. having CcdB in the cell and adding small amounts of a concentrated CcdA solution as well as vice versa. For simple binding phenomena, both experiments are expected to produce the same results. Interestingly, different starting points lead to different apparent stoichiometries and affinities (Fig. 3,A and B). Starting from an excess of CcdB, saturation is reached at a ratio CcdA:CcdB of 1:2. In the reverse case, starting from an excess of CcdA, a binding signal was recorded until a ratio of above 1:1 was reached. In both cases, but especially noticeably in the second condition that lead to an approximately 1:1 stoichiometry, the contents of the calorimetric cell was slightly turbid after the experiment. This is indicative of the formation of insoluble aggregates. The two titration thermograms show a second difference: in the first case saturation is attained rather abruptly, whereas in the second case it is reached smoothly. This indicates that the interaction is characterized by more than one binding constant and/or might be partially irreversible. A thermogram obtained from such a complex interaction cannot be analyzed with the standard software to obtain the involved microscopic binding constants. Rough estimates of the binding constants can be obtained if we assume that in the early phase of the transition (when one of the reactants is in excess) one binding constant dominates the process. Assuming a model with two microscopic binding constantsKL and KH, one constant will dominate the early stages of the forward titration, while the other will dominate in the early stages of the reverse titration. Such a model is described in detail in the following paragraph. To understand the complex behavior observed for the interaction between CcdA and CcdB, we performed numerical simulations of the forward and reverse titrations. We assumed a model in which CcdA and CcdB can form long chains in a cooperative way. Such a model is realistic given the strong tendency of CcdA and CcdB for forming precipitates when mixed in a 1:1 molar ratio and the possibility of producing a soluble CcdA2CcdB4 complex. In this cooperative interaction model, initial binding of a single dimer of CcdB2 to CcdA2 occurs with a binding constantKL. Addition of a second molecule of CcdB2 to an existing CcdA2CcdB2complex involves a higher affinity constant KH. In a similar way, higher molecular weight species are produced by addition of more CcdA2 and CcdB2 dimers using the same binding constants. The equilibrium equations of the model are given in the experimental procedures section. This cooperative model assumes a conformational change on the part of CcdA. Most likely, CcdA is partly unfolded when not bound to CcdB. Binding of a first CcdB2dimer to CcdA2 results in proper folding and creates a more stable binding site for the second CcdB2 dimer. It is not possible to obtain correct values of KLand KH from the ITC experiments. However, the applied protein concentrations and the shape of the observed titration thermograms permit us to make a reasonable estimate for the range of the binding constants involved (105m−1 <KL <KH < 108m−1) (29Weber G. Protein Interactions. Chapman & Hall, New York1992: 1-17Google Scholar). Indeed a sigmoidal titration thermogram is only observed within this range. Smaller values result in a soft featureless increase, while larger values lead to an abrupt jump (Fig. 3C). Based on the above considerations we estimated two numerical values for the binding constants: KL = 106m−1 and KH = 5 107m−1. Typical results of our simulations are given in Fig.4. We calculated the distribution of the protein into different complexes mimicking the experimental conditions of the ITC titration experiments (relatively high concentrations: 2 10−5m protein). Because of the robustness of the model the result is only marginally influenced by variations of the binding constants in the same order of magnitude. The calculation shows that at equimolar amounts of CcdA and CcdB most of CcdA is present in the form of the higher aggregates of the type (A2B2)n. Only at a molar ratio CcdB:CcdA of above ∼3:1, the soluble hexamer CcdA2CcdB4 will be the dominant form of CcdA. In Fig. 4B it can be seen that at this excess a maximal fraction of CcdB will also be in this hexameric form. Above this molar ratio most of CcdB will be in the form of the free dimer, because all the available CcdA is consumed in complexes. On the other hand at close to equimolar concentrations and at molar ratios CcdB:CcdA below 1, especially between 0.1 and 1, CcdB is found in higher aggregates and the hexamer CcdA4CcdB2. Such a situation can be characterized as an aggregation scenario, confirmed by gel filtration experiments. It is known from literature that both ccd proteins participate in the autoregulation of the system (7de Feyter R. Wallace C. Lane D. Mol. Gen. Genet. 1989; 218: 481-486Crossref PubMed Scopus (50) Google Scholar, 8Tam J.E. Kline B.C. Mol. Gen. Genet. 1989; 219: 26-32Crossref PubMed Scopus (70) Google Scholar, 9Tam J.E. Kline B.C. J. Bacteriol. 1989; 171: 2353-2360Crossref PubMed Google Scholar). Only few details on the interaction of the ccd proteins with the ccdoperator DNA are known. CcdA binds on DNA and CcdB does not (also see below). To better characterize the binding site(s) of CcdA and the role of CcdB, we examined the DNase I footprint to a 157-bp fragment (F4R1: fragment from start of F4 to end of R1; see Fig.5C). These experiments revealed a large region of interaction (∼110 bp; Fig. 5, Aand B), in agreement with previous data obtained with the lysate of a strain overexpressing the ccd proteins (9Tam J.E. Kline B.C. J. Bacteriol. 1989; 171: 2353-2360Crossref PubMed Google Scholar). Whereas Tam and Kline (9Tam J.E. Kline B.C. J. Bacteriol. 1989; 171: 2353-2360Crossref PubMed Google Scholar) only detected protection of one strand, we definitely observed a complex footprint for each strand. Protected stretches of ∼7–10 nucleotides long are separated by 3–6-nucleotide-long segments that either remained normally accessible to the nuclease or became hyper-reactive to DNase I cleavage in the presence of both Ccd proteins. These alternating patches of protection and hypersensitivity toward digestion are mostly staggered by a few nucleotides toward the 3′-end on one strand with respect to the complementary partner (Fig. 5C). Such a pattern is consistent with a series of CcdA·CcdB complexes that spiral along a 120-bp region. Footprinting experiments were performed using different CcdA:CcdB ratios ranging from 1:1 to 3:1. The observed footprint patterns are indistinguishable (Fig. 5A). In the absence of CcdB, only a slight effect but in no way a clear pattern of protection was observed (Fig. 5B). Mobility-shift experiments performed with an aliquot of the very same samples (data not shown) clearly demonstrated binding of CcdA to the operator fragment. The lack of a distinct footprint most likely reflects the formation of unstable CcdA-operator DNA complexes with high on and off rates (see below). The region protected by the CcdA·CcdB complex against DNase I cleavage contains two interesting stretches, the promoter region, and a 6-bp palindrome sequence just downstream of the −10 promoter element (Fig. 5C). To better characterize the sequence requirements for binding of the Ccd proteins and to determine the minimal target site for CcdA-CcdB binding, we have performed gel retardation experiments with a variety of DNA fragments: the 157-bp F4R1 fragment and three subfragments thereof (OP12, Prom, and Pal; for a definition of these fragments, see Fig.5C) that were synthesized according to the nucleotide sequence described by Tam and Kline (9Tam J.E. Kline B.C. J. Bacteriol. 1989; 171: 2353-2360Crossref PubMed Google Scholar). Gel shift assays indicated that CcdA retards all four of these fragments, but CcdB does not. Fig.6A shows the titration of F4R1 with CcdA. The transition from unbound to CcdA-bound F4R1 DNA is very sharp, pointing to a strong cooperative interaction. Relatively low concentrations of CcdB (around 0.5 μm) are sufficient to provide an apparent increase in affinity of CcdA for F4R1. A larger shift of the DNA fragment is observed. The affinity of CcdA for F4R1 in the presence of CcdB is estimated to be about 10 times higher than that observed in its absence as measured by the amount of CcdA necessary to produce a band shift. Similarly, we demonstrated binding of CcdA alone and the CcdB-induced increase in the apparent binding constant and "supershifting," for the 34-bp-long OP12 fragment (Fig.6C). In contrast, binding of CcdA on the 25-bp fragment containing the palindrome sequence (Pal) was only detectable in the presence of CcdB, whereas binding on the 21-bp-long promoter sequence (Prom) was hardly detectable (Fig. 6C). Combined (see also DNase I footprinting), these results indicate that the palindrome region might constitute the nucleation site for binding of multiple CcdA-CcdB molecules to the control region of the ccdoperon. These retardation experiments con
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