title: Importance of the α10 helix for DevR activation: A road map for screening inhibitors against DevR-mediated dormancy of Mycobacterium tuberculosis.
abstract: OBJECTIVE
  Bacterial persistence is the hallmark of tuberculosis (TB) and poses the biggest threat to the success of any antitubercular drug regimen. The DevR/DosR dormancy regulator of Mycobacterium tuberculosis belongs to the NarL subfamily of response regulators and is essential for M. tuberculosis persistence in macaque models of TB. The DevR/DosR crystal structure revealed a unique (αβ)4topology instead of the classical (αβ)5structure found in the receiver domain of other regulators in this subfamily. It was proposed that phosphorylation may culminate in the formation of a DNA-binding-competent dimeric species via α10-α10 helix interactions. Here, we deciphered the role of the α10 helix in activation of the DevR/DosR response regulator in M. tuberculosis.
  
  
  METHODS
  Wild-type (WT) and mutant DevR [α10-helix-deleted DevR (DevRΔα10)] proteins were cloned in suitable plasmids and expressed in Escherichia coli and M. tuberculosis strains. An in vitro phosphorylation assay was performed using acetyl phosphate, and the dimeric/oligomeric status of WT DevR and mutant proteins in the presence or absence of phosphorylation was assessed by glutaraldehyde-based in vitro cross-linking, followed by western blot analysis. Additionally, recombinant M. tuberculosis strains expressing WT and mutant DevR proteins were assessed for dormancy regulon gene expression under aerobic and hypoxic conditions by western blot analysis. An electrophoretic mobility shift assay was performed to assess the in vitro DNA-binding activity of DevR proteins to the target DNA, and biophysical characterization was performed using circular dichroism spectroscopy, fluorescence spectroscopy, and thermal shift assays.
  
  
  RESULTS
  Our results revealed that DevR structure and activity are modulated by phosphorylation-dependent α10 helix dimerization. In its hyperphosphorylated state, DevRΔα10is defective in DNA binding and exhibits an open and less stable conformation. The combined results of in vitro cross-linking and genetic analysis established an essential role for the α10 helix in postphosphorylation dimerization of DevR and gene activation. The importance of the α10 helix for dormancy regulon induction in M. tuberculosis established the α10-α10 helix interaction as a novel target in the DevR-signaling pathway for developing inhibitors against DevR, a key regulator of hypoxia-triggered dormancy.
  
  
  CONCLUSIONS
  This study established the importance of the α10 helix for DevR activation in M. tuberculosis and proposed a novel molecular tool to screen small-molecule inhibitors targeting dimerization of DevR in the absence (inactive state) or presence of phosphorylation (active state) to combat latent TB infection. This concept can be extended to screen inhibitors against response regulators where dimerization is crucial for their activation.
authors.affiliation: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India; Experimental Animal Facility, National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, India.
authors.forename: Atul
authors.initials: A
authors.lastname: Vashist
authors.affiliation: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: D
authors.initials: D
authors.lastname: Prithvi Raj
authors.affiliation: Experimental Animal Facility, National JALMA Institute of Leprosy and Other Mycobacterial Diseases, Tajganj, Agra, India.
authors.forename: Umesh Datta
authors.initials: UD
authors.lastname: Gupta
authors.affiliation: School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
authors.forename: Rajiv
authors.initials: R
authors.lastname: Bhat
authors.affiliation: Department of Biotechnology, All India Institute of Medical Sciences, New Delhi, India. Electronic address: jstyagi@aiims.ac.in.
authors.forename: Jaya Sivaswami
authors.initials: JS
authors.lastname: Tyagi
keywords: Dormancy; Hypoxia; Mycobacterium tuberculosis; Phosphorylation; Two-component signal transduction system