The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

Abstract

Cholesterol can be a major carbon source forMycobacterium tuberculosisduring infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, includingkstR, are either induced during infection or are essential for survival ofM. tuberculosis in vivo In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50for ligand = 25 nm). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain. In contrast, structures of KstR·ligand complexes are highly similar to each other and demonstrate a position of the DNA-binding domain that is unfavorable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release.

Keywords: CoA thioester; Mycobacterium tuberculosis; bacterial metabolism; cholesterol metabolism; protein structure; transcription repressor.

FIGURE 1.

Schematic of cholesterol degradation in M. tuberculosis. A–D refer to rings A–D.

FIGURE 2.

SEC-MALS analysis of KstR. KstR was eluted in a single peak as detected by refractive index detection (solid and dashed lines represent samples loaded at 2.5 and 5.0 mg/ml, respectively). Calculated molar mass across the peaks (filled and empty circles at 2.5 and 5.0 mg/ml, respectively) indicates a dimeric form of KstR in solution. The graph shows the complete elution range of the Superdex 200 10/300 GL column, and the inset shows an enlarged region around the peak.

FIGURE 3.

Analysis of the interaction of KstR with DNA and ligands. A, global fitting of SPR equilibrium response of the KstR-DNA interaction. Change in response units (RU) at equilibrium is plotted against concentration of KstR dimer. B, EMSA to identify KstR ligands. All lanes contain 0.6 μm DNA probe and 1.2 μm KstR monomer plus either 20 μm possible effector or 600 ng of nonspecific (NS) (salmon sperm) DNA. C, intrinsic fluorescence of KstR in the presence of 3OCh-CoA (circles, solid line) or 4-BNC-CoA (squares, dashed line). Samples contain 2 μm KstR monomer and 0–8 μm ligand. D, SPR analysis of 15 nm KstR dimer and immobilized DNA in the presence of 3OCh-CoA (circles, solid line) or 4-BNC-CoA (squares, dashed line). Residual binding is normalized against the binding of KstR in the absence of ligand. Shown values are the average of triplicate experiment with two readings for each repeat. Error bars indicate standard deviation for all readings.

FIGURE 4.

Structure of ligand-free KstR. A, the dimer of the form I structure with each monomer colored from blue (N terminus) to red (C terminus) and helices labeled. The LBD and DBD are indicated. B, superposition of form I (maroon) and form II (dimer 1; green) overlaid at the central four-helix bundle composed of helices α8 and α9 from each subunit.

FIGURE 5.

Structure of ligand-bound KstR. A, difference electron density in the ligand pocket of KstR indicates the presence of bound ligands. F_o − F_c electron density maps (green mesh; contoured at 2σ) were calculated prior to modeling the ligands (gray stick model) in structures of KstR determined in complex with 3OCh-CoA and 4-BNC-CoA. B, binding of 4-BNC-CoA (shown as a stick model) into the ligand-binding pocket of KstR (shown as schematic and surface representations). C, the contacts made by KstR to both 3OCh-CoA and 4-BNC-CoA. Hydrophobic contacts shown in red; hydrogen bonds are shown as green dashed lines.

FIGURE 6.

Structural changes in KstR due to ligand binding. A, superposition of monomer structures of ligand-free KstR (green; form II, dimer 1) and KstR in complex with 3OCh-CoA (blue). The movement of the DBD relative to the α8-α9 dimer interface is shown by red arrows. B, the ligand-induced movements of the KstR monomer are coordinated with a change in the conformation of Trp-164 (shown as a stick model).