Structural basis for the function of the transcriptional regulator KstR from Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb), the causative agent of human lung TB, is well-known for its strong persistence inside the human macrophage. Cholesterol catabolism has been suggested to be one of the critical metabolic processes for the phagosome-dwelling Mtb. The transcriptional regulator KstR, which controls a large cohort of genes taking part in the cholesterol degradation pathway, is a potential drug target in Mtb. In this study, KstR has been biochemically and structurally characterised as a basis for discovering new drugs targeting the cholesterol metabolism in Mtb. The interactions between KstR and cognate DNA or ligands have been investigated by multiple biochemical techniques (i.e. electrophoretic mobility shift assay, surface plasmon resonance and intrinsic fluorescence quenching assay). Protein-DNA binding assays have demonstrated that the 21-amino-acid extension at the N-terminus of KstR imposes a positive but non-essential role for KstR-DNA interaction. Protein-ligand binding assays and competitive assays (of protein, DNA and ligand) have been used to identify three 3-oxo-Δ4 steroid ligands for KstR. The strong interactions with KstR of these compounds indicate that they are endogenous ligands of KstR. In addition, the regulator has also been shown to interact with an aliphatic ligand, palmitoyl-CoA, albeit with a weaker affinity and lower specificity. The crystallographic structures of KstR in various complexation states (i.e. free, ligandbound and DNA-bound) have been solved and analysed, which provides the basis for KstR’s specificity with its ligands and cognate DNA. A structure-based allosteric mechanism is postulated from the conformational changes of KstR among these forms. In addition, the structures of the KstR-DNA complex hint at a potential role for intrinsic DNA shape (determined by its sequence) in the protein-DNA interaction, which has never been observed in other DNA-major-groove binding proteins. The study also explores an application of the fragment-based drug discovery approach on KstR. A thermal shift assay has been used to screen a fragment library, from which a subset of fragments stabilising and destabilising the protein has been identified. The crystallographic structure of KstR in complex with the stabilising fragments have been solved, which provides the background for the future drug design targeted at KstR.