Biophysical characterization and interaction study of WhiB6 protein of Mycobacterium tuberculosis with espA.

Tuberculosis is an intractable disease because of the peculiar nature of the virulent properties of Mycobacterium tuberculosis (Mtb). The probable transcriptional regulator WhiB6 protein plays a crucial role in the virulence systems of Mtb. It regulates the expression of genes essential for the virulence pathways by binding to their promoter region; espA (encoding ESX-1 secretion-associated protein EspA) is one such gene. Herein, we have used biophysical methods, including steady-state intrinsic fluorescence spectroscopy, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and surface-enhanced Raman spectroscopy (SERS), to understand the interaction of apo-WhiB6 protein with espA promoter DNA. For the first time, we report the conformational details and biophysical parameters related to the WhiB6-espA-promoter-DNA interaction. WhiB6 binds to the DNA with moderate affinity, as revealed by ITC. It is an entropy-driven process, signifying the importance of hydrophobic interaction and an increase in conformational flexibility upon binding. Addition of salt changes the binding from endothermic to exothermic, revealing the increase in electrostatic interaction between protein and DNA with concomitant decrease in flexibility. CD and SERS studies suggest subtle perturbation in the secondary conformation of the protein upon binding to the DNA. ITC titration data of an arginine-to-leucine mutant in the arginine-rich region (GRARAF) of WhiB6 suggest involvement of these residues in the binding with DNA. Preventing the binding of WhiB6 with promoter DNA of the virulence genes can hinder the functioning of Mtb and hence can act as an effective therapeutic intervention for tuberculosis.