Structural and Functional Characterization of a Ketosteroid Transcriptional Regulator of Mycobacterium tuberculosis

Catabolism of host cholesterol is critical 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 molecule, 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 resolution crystal structure of the KstR2 Mtb ·HIP-CoA complex reveals that the KstR2 Mtb dimer accommodates two molecules of HIP-CoA. Each ligand binds in an elongated cleft spanning the dimerization interface such that the HIP and CoA moieties interact with different KstR2 Mtb protomers. In isothermal titration calorimetry studies, the dimer bound 2 eq of HIP-CoA with high affinity ( K d = 80 ± 10 nm) but bound neither HIP nor CoASH. Substitution of Arg-162 or Trp-166, residues that interact, respectively, with the diphosphate and HIP moieties of HIP-CoA, dramatically decreased the affinity of KstR2 Mtb for HIP-CoA but not for its operator sequence. The variant of R162M that decreased the affinity for HIP-CoA (ΔΔ G = 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 homologue and a DNA-bound homologue 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.KstR2 regulates cholesterol catabolic genes in Mycobacterium tuberculosis . Results Dimeric KstR2 Mtb binds two molecules of HIP-CoA with high affinity. Each binding site spans the subunits and includes residues conserved in TetR family repressors (TFRs) that bind CoA thioesters. Conclusion HIP-CoA binding to KstR2 Mtb induces a conformation that abrogates DNA binding. Significance The study identifies molecular determinants of cholesterol catabolism and CoA binding in TFRs.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.