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: For crystallization trials, kpRstA DBD was mixed with 23-bp DNA substrate (Figure 2C) at a molar ratio of 2:1 (DNA:protein) and concentrated to 20 mg/ml in preparation buffer (20 mM Tris-HCl, pH 7.5 and 100 mM NaCl). kpRstA RD was similarly prepared except that the preparation buffer contained additional 5.3 mM BeSO4, 35 mM NaF and 7 mM MgCl2. Initial crystallization trials were performed with commercially available kits (Hampton Research, USA) using the hanging drop vapor diffusion method (43). Specifically, 1 μl of concentrated complex was mixed with an equal amount of reservoir solution and equilibrated against 500 μl of reservoir solution at 26°C. After optimization of crystallization conditions, rhombus-shaped kpRstA RD crystals appeared in 100 mM Bis–Tris, pH 6.5 and 25% (v/v) polyethylene glycol 3350, whereas kpRstA DBD/DNA formed plate-shaped crystals in 100 mM Bis–Tris, pH 5.5, 28% (v/v) polyethylene glycol 400 and 1.32 M sodium formate. The phase of kpRstA RD crystals were determined by single-wavelength anomalous diffraction (SAD) of SeMet-labeled crystals at 0.97874 Å (Se-peak). The phase of kpRstA DBD/DNA crystals were determined by collecting multi-wavelength anomalous diffraction (MAD) data on a single SeMet-labeled crystal of kpRstA DBD(L153M/L168M)–DNA complex at three different wavelengths: 0.97879 Å (Se-edge), 0.96357 Å (Se-remote) and 0.97862 Å (Se-peak). All data were collected on an ADSC Quantum-315 CCD area detector at beamline BL13B1 of the National Synchrotron Radiation Research Center, Hsinchu, Taiwan. X-ray diffraction data integration and scaling were performed using the HKL2000 package (44). For the MAD experiment, data from each wavelength were indexed according to the same crystal orientation matrix, but integrated and scaled independently. Diffraction data statistics are listed in Supplemental Table S2.
Affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan, ROC Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan.