Conformational stability and ligand binding properties of BldR, a member of the MarR family, from Sulfolobus solfataricus

• BldR has a high thermal stability (Td = 108.9 °C) as measured by CD and DSC.
• BldR has a high GuHCl resistance (Cm = 5.3 M at 25 °C) as measured by CD and DSC.
• ITC shows that BldR binds to benzaldehyde with a Kb = 7.5·105 M− 1 and 1:4 stoichiometry.
• The binding is entropically driven and causes a significant conformational change.
• Multidrug resistance can be addressed by comprehension of ligand binding mechanisms.

The multiple antibiotic resistance regulators (MarR) constitute a family of ligand-responsive transcriptional regulators ubiquitous among the bacterial and archaeal domains. BldR, an archaeal MarR member characterized from the hyperthermophilic crenarchaeon Sulfolobus solfataricus regulates its own expression and that of an alcohol dehydrogenase gene by binding to sequences in their promoters and responding to benzaldehyde as the effector molecule.In this study we assessed the thermodynamic stability of the protein BldR and its binding with benzaldehyde through biophysical measurements. The temperature- and denaturant-induced unfolding experiments, performed by means of circular dichroism (CD) and differential scanning calorimetry (DSC), showed that BldR has an extremely high thermal stability (Td = 108.9 °C) and a remarkable resistance against GuHCl (Cm = 5.3 M at 25 °C). The unfolding Gibbs energy, ΔdG (H2O), calculated by the linear extrapolation model from GuHCl-induced unfolding equilibrium curve, is 72.2 kJ mol− 1.ITC binding experiments showed that four benzaldehyde molecules bind to one BldR dimer with a binding constant Kb of 7.5·105 M− 1, being the binding entropically driven. ITC, CD and fluorescence results are consistent with a conformational change induced by benzaldehyde binding, further proving that this molecule is a specific effector for BldR modulating its DNA binding activity.