Functional and in-silico characterization of Mce1R of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis mostly causes latent tuberculosis infection in humans by entering in a state of "Dormancy" where it silently resides within the host cellular system without establishing active disease symptoms. During dormancy, the pahogen utilizes host derived fatty acids and cholesterol as sole sources of carbon and energy to promote survival and pathogenicity. M. tuberculosis imports fatty acids from extracellular environment through Mce1 transporter which is under transcriptional control of Mce1R, a VanR-type regulator. Mce1R deletion mutants of M. tuberculosis are unable to cause persistent infection which makes Mce1R a novel drug target for anti-tuberculosis drug discovery approaches. In this work functional and in-silico characterization of Mce1R has been performed to some extent. Mce1R gene has been cloned, expressed and purified to homogeneity. Purified Mce1R could specifically bind to the mce1 promoter DNA (operator) with moderate affinity (Kd = 0.35 ± 0.02 μM). Initially, the monomeric unit structure of Mce1R has been modeled using Phyre2 server and validated by computational and experimental methods. Since VanR type regulators form dimers, the dimeric model of Mce1R was modeled using the Galaxy Homomer server and validated again. The structure is found to carry an N-terminal unstructured arm with distinct N- and C-terminal domains like that of VanR-type regulators. Coarse grain molecular dynamics simulation suggests that the N-terminal domain including the N-terminal arm structure is more flexible while the C-terminal domain is comparatively rigid. Structure-guided sequence alignment among the structural orthologs of Mce1R revealed that the N-terminal domain of Mce1R is rich in many highly conserved residues while the C-terminal domain residues are mostly substituted by similar types of residues which suggests that structural dynamics of Mce1R is preserved among the structural orthologs. A ligand-binding cavity has been identified at the C-terminal domain of Mce1R and through binding site matching approach employed by the ProBis server; fatty acids were selected as possible ligands for Mce1R. Molecular docking followed by analysis of Mce1R fatty acids interactions reveled that several cavity residues are mediating hydrophobic interaction with the fatty acid ligands. All atom molecular dynamics simulation of the docked complexes using GROMACS suggests that ligand binding stabilizes the structure of Mce1R. Interestingly, Mce1R is found to preferably form stable complexes with long chain fatty acids and undergo distinct structural changes after binding.