Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis

With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.     

Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved β-sheet on top of a long and a short α-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k _cat of 1.1 × 10² sec⁻¹. The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.     

Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial survival during infection, has a double hotdog fold

The Mycobacterium tuberculosis genome contains about 4000 genes, of which approximately a third code for proteins of unknown function or are classified as conserved hypothetical proteins. We have determined the three-dimensional structure of one of these, the rv0216 gene product, which has been shown to be essential for M. tuberculosis growth in vivo. The structure exhibits the greatest similarity to bacterial and eukaryotic hydratases that catalyse the R-specific hydration of 2-enoyl coenzyme A. However, only part of the catalytic machinery is conserved in Rv0216 and it showed no activity for the substrate crotonyl-CoA. The structure of Rv0216 allows us to assign new functional annotations to a family of seven other M. tuberculosis proteins, a number if which are essential for bacterial survival during infection and growth.     

Solution structure of the guanine nucleotide-binding STAS domain of SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis

The structure and intrinsic activities of conserved STAS domains of the ubiquitous SulP/SLC26 anion transporter superfamily have until recently remained unknown. Here we report the heteronuclear, multidimensional NMR spectroscopy solution structure of the STAS domain from the SulP/SLC26 putative anion transporter Rv1739c of Mycobacterium tuberculosis. The 0.87-Å root mean square deviation structure revealed a four-stranded β-sheet with five interspersed α-helices, resembling the anti-σ factor antagonist fold. Rv1739c STAS was shown to be a guanine nucleotide-binding protein, as revealed by nucleotide-dependent quench of intrinsic STAS fluorescence and photoaffinity labeling. NMR chemical shift perturbation analysis partnered with in silico docking calculations identified solvent-exposed STAS residues involved in nucleotide binding. Rv1739c STAS was not an in vitro substrate of mycobacterial kinases or anti-σ factors. These results demonstrate that Rv1739c STAS binds guanine nucleotides at physiological concentrations and undergoes a ligand-induced conformational change but, unlike anti-σ factor antagonists, may not mediate signals via phosphorylation.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Crystal structure of a conserved hypothetical protein from Escherichia coli

The crystal structure of a conserved hypothetical protein from Escherichia coli has been determined using X-ray crystallography. The protein belongs to the Cluster of Orthologous Group COG1553 (National Center for Biotechnology Information database, NLM, NIH), for which there was no structural information available until now. Structural homology search with DALI algorism indicated that this protein has a new fold with no obvious similarity to those of other proteins with known three-dimensional structures. The protein quaternary structure consists of a dimer of trimers, which makes a characteristic cylinder shape. There is a large closed cavity with approximate dimensions of 16 Å × 16 Å × 20 Å in the center of the hexameric structure. Six putative active sites are positioned along the equatorial surface of the hexamer. There are several highly conserved residues including two possible functional cysteines in the putative active site. The possible molecular function of the protein is discussed.     

Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis

Rv3868, a conserved hypothetical protein of the ESAT-6 secretion system of Mycobacterium tuberculosis, is essential for the secretion of at least four virulence factors. Each protein chain is approximately 63 kDa and assembles into a hexamer. Limited proteolysis demonstrates that it consists of two domains joined by a linker. The N-terminal domain is a compact, helical domain of approximately 30 kDa and apparently functions to regulate the ATPase activity of the C-terminal domain and the oligomerization. The nucleotide binding site is situated in the C-terminal domain, which exhibits ATP-dependent self-association. It is also the oligomerization domain. Dynamic fluorescence quenching studies demonstrate that the domain is proximal to the C terminus in the apoprotein and exhibits a specific movement upon ATP binding. In silico modeling of the domains suggests that Arg-429 of a neighboring subunit forms a part of the binding site upon oligomerization. Mutational analysis of binding site residues demonstrates that the Arg-429 functions as the important "sensor arginine" in AAA-ATPases. Protein NMR experiments involving CFP-10 and activity assays rule out a general chaperone-like function for Rv3868. On the other hand, ATP-dependent "open-close" movements of the individual domains apparently enable it to interact and transfer energy to co-proteins in the ESX-1 pathway.     

Rv0802c acetyltransferase from Mycobacterium tuberculosis H37Rv

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.     

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-l-methionine (AdoMet) has been determined at 1.98 A resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a beta-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively. Copyright 12001 Academic Press.     

结核分枝杆菌蛋白Rv3425对细菌表型及毒力影响的研究

为探索蛋白Rv3425在结核分枝杆菌(Mycobacterium tuberculosis,M. tuberculosis)中的功能,本研究以耻垢分枝杆菌(Mycobacterium smegmatis,M. smegmatis)为模式菌株,构建重组了耻垢分枝杆菌Ms-Rv3425。分别将构建菌株(Ms-Rv3425)、野生株(Ms)及空载对照(Ms-Pact)接种于7H9-OADC培养基中37 ℃培养,观察Ms-Rv3425与Ms及Ms-Pact之间在生长速率、菌落形态、生物膜以及聚集度方面的差异。分别用低pH值以及含有十二烷基磺酸钠(sodium dodecyl sulfate,SDS)、氨苄西林、异烟肼及利福平的培养基进行培养,计算存活率以分析抗逆和抗药能力;用上述压力条件培养结核分枝杆菌标准株H37Ra,分析Rv3425内源表达量的变化;进行THP-1细胞感染和BALB/c小鼠攻毒实验分析菌株的毒性变化。结果显示,与Ms及Ms-Pact相比,Ms-Rv3425的菌落形态更为粗糙且隆起,成膜及聚集能力增强;在压力条件下,Ms-Rv3425表现出更高的抗逆和抗药能力,H37Ra中Rv3425的表达量也显著上调;胞内存活率及小鼠致死率更高,各脏器病理损伤更为严重。综上所述,过表达Rv3425能够改变耻垢分枝杆菌的表型,提高抗逆性、抗药性和毒力。深入探讨PPE家族蛋白Rv3425的功能,将为结核病的防治带来新的视角。     

Structural characterization of the multidomain regulatory protein Rv1364c from Mycobacterium tuberculosis

The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σ(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σ(F) like) domain and?a C-terminal anti-σ(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6??) and a phosphatase/kinase dual domain construct (at 2.6??) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding,?affects the accessibility of the serine of the C-terminal, anti-σ(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σ(F).     

Crystal structure of a novel esterase Rv0045c from Mycobacterium tuberculosis

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 α/β hydrolase fold family. The structure of esterase Rv0045c contains two distinct domains: the α/β 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.