title: Structural and functional characterization of a ketosteroid transcriptional regulator of Mycobacterium tuberculosis.
abstract: 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 (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 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.
authors.affiliation: From the Departments of Biochemistry and Molecular Biology and.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Crowe
authors.affiliation: the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto M5S 3E5, Canada, and.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Stogios
authors.affiliation: Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver V6T 1Z3, Canada.
authors.forename: Israël
authors.initials: I
authors.lastname: Casabon
authors.affiliation: the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto M5S 3E5, Canada, and The Midwest Center for Structural Genomics (MCSG), Argonne National Laboratory, Argonne, Illinois 60439.
authors.forename: Elena
authors.initials: E
authors.lastname: Evdokimova
authors.affiliation: the Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto M5S 3E5, Canada, and The Midwest Center for Structural Genomics (MCSG), Argonne National Laboratory, Argonne, Illinois 60439 alexei.savchenko@utoronto.ca.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.affiliation: From the Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, Life Sciences Institute, The University of British Columbia, Vancouver V6T 1Z3, Canada, leltis@mail.ubc.ca.
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D003065:Coenzyme A / Q000737:chemistry; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007664:Ketosteroids / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000235:genetics* / Q000472:pathogenicity; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins / Q000235:genetics*; D013753:Tetracycline Resistance / Q000235:genetics; D014376:Tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D007664:Ketosteroids; D008024:Ligands; D012097:Repressor Proteins; D002784:Cholesterol; D003065:Coenzyme A
keywords: Actinobacteria; Bacterial Metabolism; Cholesterol; CoA Thioester; Mycobacterium Tuberculosis; Steroid Catabolism; Transcription Repressor