ABSTRACT
Protein kinases play central roles in the survival of Mycobacterium tuberculosis within host. Here we report the individual high-resolution crystal structures of the sensor domain (in both monomer and dimer forms) and the kinase domain of PknI, a transmembrane protein member of the serine/threonine protein kinases (STPKs) family. PknI is the first STPK identified whose sensor domain exists in a monomer-dimer equilibrium. Inspection of the two structures of the sensor domain (PknI_SD) revealed conformational changes upon dimerization, with an arm region of critical importance for dimer formation identified. Rapamycin-induced dimerization of unphosphorylated fusions of PknI juxtamembrane and the kinase domain, intended to mimic the dimerization effect presumably imposed by PknI_SD, was observed to be able to activate auto-phosphorylation activity of the kinase domain. In vivo experiments using an M. bovis model suggested PknI functions as a dimer in the regulation of M. tuberculosis growth.
Subject(s)
Bacterial Proteins/chemistry , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/enzymology , Protein Domains , Protein Serine-Threonine Kinases/metabolismABSTRACT
Rv1220c from Mycobacterium tuberculosis is annotated as an O-methyltransferase (MtbOMT). Currently, no structural information is available for this protein. Here, the crystal structure of MtbOMT refined to 2.0â Å resolution is described. The structure reveals the presence of a methyltransferase fold and shows clear electron density for one molecule of S-adenosylmethionine (SAM), which was apparently bound by the protein during its production in Escherichia coli. Although the overall structure of MtbOMT resembles the structures of O-methyltransferases from Cornybacterium glutamicum, Coxiella burnetti and Alfa alfa, differences are observed in the residues that make up the active site. Notably, substitution of Asp by His164 seems to abrogate metal binding by MtbOMT. A putative catalytic His-Asp pair located in the vicinity of SAM is absolutely conserved in MtbOMT homologues from all species of Mycobacterium, suggesting a conserved function for this protein.
Subject(s)
Bacterial Proteins/chemistry , Methyltransferases/chemistry , Mycobacterium tuberculosis/chemistry , S-Adenosylmethionine/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Cloning, Molecular , Corynebacterium glutamicum/chemistry , Corynebacterium glutamicum/enzymology , Coxiella burnetii/chemistry , Coxiella burnetii/enzymology , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Medicago sativa/chemistry , Medicago sativa/enzymology , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Mycobacterium tuberculosis/enzymology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/metabolism , Structural Homology, Protein , Substrate SpecificityABSTRACT
Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 Å resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 Å resolution. The crystals belonged to space group C2, with unit-cell parameters a = 122.0, b = 47.1, c = 89.5 Å, α = γ = 90, ß = 124.5°. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 Å resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.
