ABSTRACT
Polygalacturonases are pectate-degrading enzymes that belong to glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond between neighboring galacturonasyl residues of the homogalacturonan substrate. The acidic polygalacturonase PehA from Agrobacterium vitis was overexpressed in Escherichia coli, where it accumulated in the periplasmic fraction. It was purified to homogeneity via a two-step chromatography procedure and crystallized using the hanging-drop vapour-diffusion technique. PehA crystals belonged to space group P2(1), with unit-cell parameters a = 52.387, b = 62.738, c = 149.165 A, beta = 89.98 degrees . Crystals diffracted to 1.59 A resolution and contained two molecules per asymmetric unit. An initial structure determination by molecular replacement indicated a right-handed parallel beta-helix fold.
Subject(s)
Bacterial Proteins/chemistry , Polygalacturonase/chemistry , Rhizobium/enzymology , X-Ray Diffraction , Bacterial Proteins/genetics , Crystallization/methods , Polygalacturonase/genetics , Rhizobium/geneticsABSTRACT
Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233-Asp242). Residues Leu186-Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined alpha-helix and beta-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions.
Subject(s)
Active Transport, Cell Nucleus , Bacterial Toxins/chemistry , Arginine/chemistry , Binding Sites , Crystallography, X-Ray , Escherichia coli/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, SecondaryABSTRACT
Cytolethal distending toxin (CDT) is a secreted protein toxin produced by several bacterial pathogens. The biologically active CDT subunit CdtB is an active homolog of mammalian type I DNase. Internalization of CdtB and subsequent translocation into the nucleus of target cells results in DNA-strand breaks, leading to cell-cycle arrest and apoptosis. CdtB crystals were grown using microbatch methods with polyethylene glycol 8000 as the precipitant. The CdtB crystals contain one molecule of MW 30.5 kDa per asymmetric unit, belong to space group P2(1)2(1)2(1) and diffract to 1.72 A.
Subject(s)
Bacterial Toxins/chemistry , Escherichia coli Proteins/chemistry , Crystallization , Crystallography, X-RayABSTRACT
Phosphatidylinositol transfer protein (PITP) is a ubiquitous eukaryotic protein that preferentially binds either phosphatidylinositol or phosphatidylcholine and catalyzes the exchange of these lipids between membranes. Mammalian cytosolic PITPs include the ubiquitously expressed PITPalpha and PITPbeta isoforms (269-270 residues). The crystal structure of rat PITPbeta complexed to dioleoylphosphatidylcholine was determined to 2.18 A resolution with molecular replacement using rat PITPalpha (77% sequence identify) as the phasing model. A structure comparison of the alpha and beta isoforms reveals minimal differences in protein conformation, differences in acyl conformation in the two isoforms, and remarkable conservation of solvent structure around the bound lipid. A comparison of transfer activity by human and rat PITPs, using small unilamellar vesicles with carefully controlled phospholipid composition, indicates that the beta isoforms have minimal differences in transfer preference between PtdIns and PtdCho when donor vesicles contain predominantly PtdCho. When PtdCho and PtdIns are present in equivalent concentrations in donor vesicles, PtdIns transfer occurs at approximately 3-fold the rate of PtdCho. The rat PITPbeta isoform clearly has the most diminished transfer rate of the four proteins studied. With the two rat isoforms, site-directed mutations of two locations within the lipid binding cavity that possess differing biochemical properties were characterized: I84alpha/F83beta and F225alpha/L224beta. The 225/224 locus is more critical in determining substrate specificity. Following the mutation of this locus to the other amino acid, the PtdCho transfer specific activity became PITPalpha (F225L) approximately PITPbeta and PITPbeta (L224F) approximately PITPalpha. The 225alpha/224beta locus plays a modest role in the specificity of both isoforms toward CerPCho.
Subject(s)
Phosphatidylcholines/chemistry , Phospholipid Transfer Proteins/chemistry , Phospholipids/metabolism , Amino Acid Sequence , Animals , Conserved Sequence , Crystallography, X-Ray , Humans , Models, Molecular , Mutagenesis, Site-Directed , Phosphatidylcholines/metabolism , Phospholipid Transfer Proteins/genetics , Phospholipid Transfer Proteins/metabolism , Phospholipids/chemistry , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , RatsABSTRACT
Pectate lyase A (PelA) is a pectate-degrading enzyme secreted by plant pathogens. PelA from Erwinia chrysanthemi has 61% amino-acid identity and a conserved structural similarity to pectate lyase E (PelE). Although similar in structure and sequence, the enzymatic characteristics of PelA differ from those for PelE. A structural alignment of PelA and PelE reveals differences in the T1.5 loop. The sequence of the T1.5 loop in PelA was mutated to the homologous sequence in PelE. The crystal structure of the PelA T1.5 mutant has been solved to 1.6 and 2.9 A resolution. The enzymatic and structural properties of the T1.5 mutant are discussed.
Subject(s)
Dickeya chrysanthemi/enzymology , Mutation , Polysaccharide-Lyases/chemistry , Bacterial Proteins/chemistry , Binding Sites , Cloning, Molecular , Crystallization , Hydrogen-Ion Concentration , Polysaccharide-Lyases/genetics , Polysaccharide-Lyases/isolation & purification , Protein Conformation , Sequence Alignment , X-Ray DiffractionABSTRACT
Pectate lyase A is a virulence factor secreted by the plant-pathogenic bacteria Erwinia chrysanthemi. The enzyme cleaves the glycosidic bond of pectate polymers by a calcium-dependent beta-elimination mechanism. The crystal structure of pectate lyase A from E. chrysanthemi EC16 has been determined in two crystal forms, monoclinic C2 to 1.8 A and rhombohedral R3 to 2.1 A. The protein structure is compared with two other pectate lyase isoforms from E. chrysanthemi EC16, pectate lyase C and pectate lyase E. Pectate lyase A is unique as it is the only acidic pectate lyase and has end products that are significantly more varied in length in comparison to those of the other four major pectate lyase isozymes. Differences and similarities in polypeptide trace, size and volume of the active-site groove and surface electrostatics are discussed.
