Crystal Structure of Mycobacterium tuberculosis Elongation Factor G1.
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ABSTRACT:
SUBMITTER: Gao X
PROVIDER: S-EPMC8446442 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.
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Project description:Translation elongation factor P (EF-P) stimulates ribosomal peptidyltransferase activity. EF-P is conserved in bacteria and is essential for cell viability. Eukarya and Archaea have an EF-P homologue, eukaryotic initiation factor 5A (eIF-5A). In the present study, we determined the crystal structure of EF-P from Thermus thermophilus HB8 at a 1.65-A resolution. EF-P consists of three beta-barrel domains (I, II, and III), whereas eIF-5A has only two domains (N and C domains). Domain I of EF-P is topologically the same as the N domain of eIF-5A. On the other hand, EF-P domains II and III share the same topology as that of the eIF-5A C domain, indicating that domains II and III arose by duplication. Intriguingly, the N-terminal half of domain II and the C-terminal half of domain III of EF-P have sequence homologies to the N- and C-terminal halves, respectively, of the eIF-5A C domain. The three domains of EF-P are arranged in an "L" shape, with 65- and 53-A-long arms at an angle of 95 degrees, which is reminiscent of tRNA. Furthermore, most of the EF-P protein surface is negatively charged. Therefore, EF-P mimics the tRNA shape but uses domain topologies different from those of the known tRNA-mimicry translation factors. Domain I of EF-P has a conserved positive charge at its tip, like the eIF-5A N domain.
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Project description:The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in ?-oxidation (?100 genes). In a previous study, the enzymology of the M. tuberculosis L-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1?Å resolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD+-bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA-FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database.
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Project description:The Rv1217c-Rv1218c multidrug efflux system, which belongs to the ATP-binding cassette superfamily, recognizes and actively extrudes a variety of structurally unrelated toxic chemicals and mediates the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis. The expression of Rv1217c-Rv1218c is controlled by the TetR-like transcriptional regulator Rv1219c, which is encoded by a gene immediately upstream of rv1218c. To elucidate the structural basis of Rv1219c regulation, we have determined the crystal structure of Rv1219c, which reveals a dimeric two-domain molecule with an entirely helical architecture similar to members of the TetR family of transcriptional regulators. The N-terminal domains of the Rv1219c dimer are separated by a large center-to-center distance of 64 Å. The C-terminal domain of each protomer possesses a large cavity. Docking of small compounds to Rv1219c suggests that this large cavity forms a multidrug binding pocket, which can accommodate a variety of structurally unrelated antimicrobial agents. The internal wall of the multidrug binding site is surrounded by seven aromatic residues, indicating that drug binding may be governed by aromatic stacking interactions. In addition, fluorescence polarization reveals that Rv1219c binds drugs in the micromolar range.
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Project description:Recent work demonstrates that the MmpL (mycobacterial membrane protein large) transporters are dedicated to the export of mycobacterial lipids for cell wall biosynthesis. An MmpL transporter frequently works with an accessory protein, belonging to the MmpS (mycobacterial membrane protein small) family, to transport these key virulence factors. One such efflux system in Mycobacterium tuberculosis is the MmpS5-MmpL5 transporter. The expression of MmpS5-MmpL5 is controlled by the MarR-like transcriptional regulator Rv0678, whose open reading frame is located downstream of the mmpS5-mmpL5 operon. To elucidate the structural basis of Rv0678 regulation, we have determined the crystal structure of this regulator, to 1.64 Å resolution, revealing a dimeric two-domain molecule with an architecture similar to members of the MarR family of transcriptional regulators. Rv0678 is distinct from other MarR regulators in that its DNA-binding and dimerization domains are clustered together. These two domains seemingly cooperate to bind an inducing ligand that we identified as 2-stearoylglycerol, which is a fatty acid glycerol ester. The structure also suggests that the conformational change leading to substrate-mediated derepression is primarily caused by a rigid body rotational motion of the entire DNA-binding domain of the regulator toward the dimerization domain. This movement results in a conformational state that is incompatible with DNA binding. We demonstrate using electrophoretic mobility shift assays that Rv0678 binds to the mmpS5-mmpL5, mmpS4-mmpL4, and the mmpS2-mmpL2 promoters. Binding by Rv0678 was reversed upon the addition of the ligand. These findings provide new insight into the mechanisms of gene regulation in the MarR family of regulators.
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Project description:There are at least 250 enzymes in Mycobacterium tuberculosis (M. tuberculosis) involved in lipid metabolism. Some of the enzymes are required for bacterial survival and full virulence. The esterase Rv0045c shares little amino acid sequence similarity with other members of the esterase/lipase family. Here, we report the 3D structure of Rv0045c. Our studies demonstrated that Rv0045c is a novel member of ?/? hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the ?/? fold domain and the cap domain. The active site of esterase Rv0045c is highly conserved and comprised of two residues: Ser154 and His309. We proposed that Rv0045c probably employs two kinds of enzymatic mechanisms when hydrolyzing C-O ester bonds within substrates. The structure provides insight into the hydrolysis mechanism of the C-O ester bond, and will be helpful in understanding the ester/lipid metabolism in M. tuberculosis.
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Project description:In bacteria, the majority of exported proteins are translocated by the Sec system, which recognizes the signal sequence of a preprotein and uses ATP and the proton motive force to mediate protein translocation across the cytoplasmic membrane. SecA is an essential protein component of this system, containing the molecular motor that facilitates translocation. Here we report the three-dimensional structure of the SecA protein of Mycobacterium tuberculosis. Each subunit of the homodimer contains a "motor" domain and a translocation domain. The structure predicts that SecA can interact with the SecYEG pore and function as a molecular ratchet that uses ATP hydrolysis for physical movement of the preprotein. Knowledge of this structure provides a framework for further elucidation of the translocation process.
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