Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element.
Bottom Line: The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion.Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner.Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.
Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.Show MeSH
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Mentions: The binding affinities of kpRstA DBD to the upstream half-site of the RstA box (DNA-16a), downstream half-site of the RstA box (DNA-16b) and the full-length RstA box (DNA-22) were studied by isothermal titration calorimetry (Figure 1). As shown on Figure 1B and Table 1, the titration curve of kpRstA DBD to DNA-16a could be fitted well with a single binding site of KD = 8.17 ± 0.54 μM with ΔH = −10.12 ± 0.12 kcal/mol and ΔS = −10.7 cal/mol/deg, indicating that the binding is enthalpically driven. In contrast, the binding of kpRstA DBD to DNA-16b did not generate enough heat to obtain reliable thermodynamic parameters, suggesting extremely weak protein–DNA interactions (Figure 1C). The titration curve of kpRstA DBD to the full length RstA box (DNA-22) was fitted with a sequential binding model of KD1 = 10.3 ± 1.13 μM, ΔH1 = −9.48 ± 0.23 kcal/mol, ΔS1 = −8.98 cal/mol/deg and KD2 = 5.15 ± 0.26 μM, ΔH2 = −6.89 ± 0.25 kcal/mol, ΔS2 = 1.07 cal/mol/deg (Figure 1D). Although the data can be fit equally well with two independent equivalent sites of KD = 3.40 ± 0.33 μM, ΔH = −7.95 ± 0.08 kcal/mol, ΔS = −1.63 cal/mol/deg, the results would not agree with the half-site binding results which showed that the downstream half-site binds to DBD poorly. The results suggest that binding of kpRstA DBD to the first site enhances the binding of the second kpRstA DBD to the second site. Mutation of Arg207, one of the key residues responsible for DNA binding (see Figure 4C), to Ala completely abolished the DNA binding of kpRstA DBD (Figure 1E).
Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.