Structural and functional characterization of a ketosteroid transcriptional regulator of mycobacterium tuberculosis


Catabolism of host cholesterol is crit. to the virulence of Mycobacterium tuberculosis and is a potential target for novel therapeutics. KstR2, a TetR family repressor (TFR), regulates the expression of 15 genes encoding enzymes that catabolize the last half of the cholesterol mol., represented by 3aα-H-4α(3’-propanoate)-7aβ-methylhexahydro-1,5-indane-dione (HIP). Binding of KstR2 to its operator sequences is relieved upon binding of HIP-CoA. A 1.6-Å resoln. crystal structure of the KstR2Mtb·HIP-CoA complex reveals that the KstR2Mtb dimer accommodates two mols. of HIP-CoA. Each ligand binds in an elongated cleft spanning the dimerization interface such that the HIP and CoA moieties interact with different KstR2Mtb protomers. In isothermal titrn. calorimetry studies, the dimer bound 2 equiv of HIP-CoA with high affinity (Kd = 80 ± 10 nm) but bound neither HIP nor CoASH. Substitution of Arg-162 or Trp-166, residues that interact, resp., with the diphosphate and HIP moieties of HIP-CoA, dramatically decreased the affinity of KstR2Mtb for HIP-CoA but not for its operator sequence. The variant of R162M that decreased the affinity for HIP-CoA (ΔΔG = 13 kJ mol-1) is consistent with the loss of three hydrogen bonds as indicated in the structural data. A 24-bp operator sequence bound two dimers of KstR2. Structural comparisons with a ligand-free rhodococcal homolog and a DNA-bound homolog suggest that HIP-CoA induces conformational changes of the DNA-binding domains of the dimer that preclude their proper positioning in the major groove of DNA. The results provide insight into KstR2-mediated regulation of expression of steroid catabolic genes and the determinants of ligand binding in TFRs. [on SciFinder(R)]

J Biol Chem