Structural and functional basis of transcriptional regulation by TetR family protein CprB from S. coelicolor A3(2).
Bottom Line: Binding of the DNA results in the restructuring of the dimeric interface of CprB, inducing a pendulum-like motion of the helix-turn-helix motif that inserts into the major groove.Experiments performed on a subset of DNA sequences from Streptomyces coelicolor A3(2) suggest that CprB is most likely a pleiotropic regulator.Apart from serving as an autoregulator, it is potentially a part of a network of proteins that modulates the γ-butyrolactone synthesis and antibiotic regulation pathways in S. coelicolor A3(2).
Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, Maharashtra, India IITB-Monash Research Academy, Mumbai 400076, Maharashtra, India.Show MeSH
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Mentions: The thermodynamic parameters for the formation of the CprB–CS and the CprB–OPB complexes were determined by performing ITC experiments. As depicted in Figure 5A, the titration of 20 μM CprB to 120 μM of CS shows a complex formation with an exothermic heat of binding. As the complex formation progressed, an incremental heat release was observed as shown in the lower panel of the binding isotherm (Figure 5A). Using an equilibrium-binding model, the data acquired were fit using one set of sites model and the Kd as well as other thermodynamic parameters like ΔH, ΔS and ΔG were obtained. The enthalpy and the entropy values for the formation of CprB–CS complex are ΔH = –10.1 kcal/mol, ΔS = –2.7 cal/mol/deg, respectively (Figure 5A). The large value of ΔH suggests that the interaction between protein and DNA is highly enthalpy driven. The formation of non-covalent interactions between the two interacting molecules is assumed to be the major contribution toward the affinity. The Kd of CprB with CS is 200 nM and the change in Gibbs free energy for this reaction is ΔG = –9.27 kcal/mol. In contrast to CprB–CS complex, the thermodynamic data obtained for CprB–OPB interaction were fitted using two sets of sites model with Kd1 = 330 nM and Kd2 = 33 nM (Figure 5B). The binding model consistent with this observation is supported by an initial binding of one of the CprB dimers to the OPB sequence, which is most likely an enthalpy-driven process. This is followed by the binding of the second dimeric unit of CprB that is most likely an entropy-driven step (Figure 5B). The values of the Kd in both cases, CS and the OPB sequence, are in agreement with the results of the DNA retardation assays. In the latter case, since the particulars of the binding mechanism cannot be observed, the values obtained by EMSA are closer to an average of the two binding constants calculated via ITC.
Affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, Maharashtra, India IITB-Monash Research Academy, Mumbai 400076, Maharashtra, India.