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.

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.

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.

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.

Eukaryotic phosphatidylinositol transfer protein is a ubiquitous multifunctional protein that transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking. The three-dimensional structure of the alpha -isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phosphatidylcholine, one of its physiological ligands, has been determined to 2.2 Angstrom resolution by x-ray diffraction techniques. A single beta -sheet and several long alpha -helices define an enclosed internal cavity in which a single molecule of the phospholipid is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface. Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases.

This study was undertaken to examine the mechanistic significance of two highly conserved residues positioned in the active site of pyruvate dehydrogenase kinase, Glu-243 and His-239. We used site-directed mutagenesis to convert Glu-243 to Ale, Asp, or Gin and His-239 to Ala. The resulting mutant kinases demonstrated a greatly reduced capacity for phosphorylation of pyruvate dehydrogenase, The Glu-243 to Asp mutant had similar to2% residual activity, whereas the Glu-243 to Ala or Gin mutants exhibited less than 0.5 and 0.1% residual activity, respectively. Activity of the His-239 to Ala mutant was decreased by similar to 90%. Active-site titration with [alpha-P-32]ATP revealed that neither Glu-243 nor His-239 mutations affected nucleotide binding. All mutant kinases showed similar or even somewhat greater affinity than the wild-type kinase toward the protein substrate, pyruvate dehydrogenase complex. Furthermore, neither of the mutations affected the inter-subunit interactions. Finally, pyruvate dehydrogenase kinase was found to possess a weak ATP hydrolytic activity, which required Glu-243 and His-239 similar to the kinase activity. Based on these observations, we propose a mechanism according to which the invariant glutamate residue (Glu-243) acts as a general base catalyst, which activates the hydroxyl group on a serine residue of the protein substrate for direct attack on the gamma phosphate. The glutamate residue in turn might be further polarized through interaction with the neighboring histidine residue (His-239).

Pectate lyase A is secreted by Erwinia chrysanthemi and is a virulence factor for soft rot diseases in plants. Crystals of pectate lyase A were obtained by vapor-diffusion techniques in the presence of polyethylene glycol. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 48.96, b = 148.86, c = 78.61 A, beta = 97.32 degrees. The crystals contain two protein molecules of 38 kDa per asymmetric unit and diffract to 2.4 A using Cu Kalpha radiation.

Phosphatidylinositol-transfer protein (PITP) is a soluble, ubiquitously expressed, highly conserved protein encoded by two genes in humans, rodents and other mammals. A cDNA encoding the alpha isoform of the rat gene was expressed to high levels in Escherichia coli, the protein purified and the homogeneous protein used for crystallization studies. Crystals of rat PITP-α were obtained by vapor-diffusion techniques using the sitting-drop method. Crystals grow within two weeks by vapor-diffusion techniques in the presence of polyethylene glycol 4000. Both crystal forms pack in the monoclinic space group P21. Crystal form I has unit-cell parameters a = 44.75, b = 74.25, c = 48.32 Å and β = 114.14°. Unit-cell parameters for crystal form II are a = 47.86, b = 73.59, c = 80.49 Å and β = 98.54°. Crystal form I has a Vm of 2.295 Å3 Da−1 and an estimated solvent content of 46.4% with one molecule per asymmetric unit, while crystal form II has a Vm of 2.196 Å3 Da−1 and an estimated solvent content of 44.0%, assuming two molecules per asymmetric unit.

The crystal structure of pectate lyase E (PelE; EC 4.2.2.2) from the enterobacteria Erwinia chrysanthemi has been refined by molecular dynamics techniques to a resolution of 2.2 A and an R factor (an agreement factor between observed structure factor amplitudes) of 16.1%. The final model consists of all 355 amino acids and 157 water molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.721[deg] for bond angles. The structure of PelE bound to a lanthanum ion, which inhibits the enzymatic activity, has also been refined and compared to the metal-free protein. In addition, the structures of pectate lyase C (PelC) in the presence and absence of a lutetium ion have been refined further using an improved algorithm for identifying waters and other solvent molecules. The two putative active site regions of PelE have been compared to those in the refined structure of PelC. The analysis of the atomic details of PelE and PelC in the presence and absence of lanthanide ions provides insight into the enzymatic mechanism of pectate lyases.

We present here preliminary diffraction data of the regulated fonn of tl1e chicken Src Protein Tvrosine Kinase.The crystals difl:i'act beyond 2.7 A witl1low mosaicity ( ~0.2') at room temperatme using syncrotron

Background: The bacterial elongation factor EF-Tu recognizes and transports aminoacyl-tRNAs to mRNA-programmed ribosomes. EF-Tu shares many structural and functional properties with other GTPases whose conformations are regulated by guanine nucleotides.Results: An intact form of Escherichia coli EF-Tu complexed with GDP has been crystallized in the presence of the EF-Tu-specific antibiotic GE2270 A, The three-dimensional structure has been solved by X-ray diffraction analysis and refined to a final crystallographic R factor of 17.2% at a resolution of 2.5 Angstrom. The location of the GE2270 A antibiotic-binding site could not be identified.Conclusions: The structure of EF-Tu-GDP is nearly identical to that of a trypsin-modified form of EF-Tu-GDP, demonstrating conclusively that the protease treatment had not altered any essential structural features. The present structure represents the first view of an ordered Switch I region in EF-Tu-GDP and reveals similarities with two other GTPases complexed with GDP: Ran and ADP-ribosylation factor-1, A comparison of the Switch I regions of the GTP and GDP forms of EF-Tu also reveals that a segment, six amino acids in length, completely converts from an a helix in the GTP complex to beta secondary structure in the GDP form. The alpha to beta switch in EF-Tu may represent a prototypical activation mechanism for other protein families.

The crystal structure of pectate lyase C (EC 4.2.2.2) from the enterobacterium Erwinia chrysanthemi (PelC) has been refined by molecular dynamics techniques to a resolution of 2.2 A to an R factor of 17.97%. The final model consists of 352 of the total 353 amino acids and 114 solvent molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.768[deg] for bond angles. The structure of PelC bound to the lanthanide ion lutetium, used as a calcium analog, has also been refined. Lutetium inhibits the enzymatic activity of the protein, and in the PelC-lutetium structure, the ion binds in the putative calcium-binding site. Five side-chain atoms form ligands to the lutetium ion. An analysis of the atomic-level model of the two protein structures reveals possible implications for the enzymatic mechanism of the enzyme.

Crystals of the pectolytic protein, polygalacturonase A, have been obtained from polyethylene glycol 8000 using vapor diffusion methods. The 52.4 kDa protein is secreted by the plant pathogenic bacteria Pseudomonas solanacearum, and is important in the virulence of this plant pathogen. The protein crystallizes in space group P2(1) and has unit-cell parameters of a = 101.9, b = 124.6, c = 48.1 A, and beta= 105 degrees 50'. The crystal has two molecules in the asymmetric unit, and diffracts maximally to a resolution of 2.1 A.

Pectate lyases are secreted virulence factors which degrade the pectate component of plant cell walls. The evolutionary-based multiple alignment of extracellular pectate lyases has been corrected using three-dimensional structural information derived from Erwinia chyrsanthemi pectate lyases C and E. The new multiple alignment reveals invariant amino acids likely to be involved in two different enzymatic functions.