4w97

Publication Abstract from PubMed

Catabolism of host cholesterol is critical to the virulence of Mycobacterium tuberculosis and 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 3aalpha-H-4alpha(3'-propanoate)-7abeta-methylhexahydro-1,5-indane-dione (HIP). Binding of KstR2 to its operator sequences is relieved upon binding of HIP-CoA. We report here a 1.6 A resolution crystal structure of the KstR2Mtb.HIP-CoA complex. In this structure, the KstR2Mtb 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 KstR2Mtb protomers. In isothermal titration calorimetry studies, the dimer bound two equivalents of HIP-CoA with high affinity (Kd = 80+/-10 nM) but did not detectably bind HIP or CoASH. Substitution of Arg-162 or Trp-166, residues that interact respectively with the diphosphate and HIP moieties of HIP-CoA in KstR2Mtb.HIP-CoA, dramatically decreased the protein's affinity for HIP-CoA but not for its operator sequence. The R162M variant's decreased affinity for HIP-CoA (DeltaDeltaG = 13 kJ/mol) is consistent with the loss of three hydrogen bonds as indicated in the structural data. Comparative structural analysis with a ligand-free rhodococcal homologue indicates that binding of 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.

Structural and functional characterization of a ketosteroid transcriptional regulator of Mycobacterium tuberculosis.,Crowe AM, Stogios PJ, Casabon I, Evdokimova E, Savchenko A, Eltis LD J Biol Chem. 2014 Nov 18. pii: jbc.M114.607481. PMID:25406313^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.