Cloning, sequence and crystallographic structure of recombinant iron superoxide dismutase from Pseudomonas ovalis.

The gene encoding the iron-dependent superoxide dismutase from Pseudomonas ovalis was cloned from a genomic library and sequenced. The ORF differs from the previously published protein sequence, which was used for the original structure determination, at 16 positions. The differences include three additional inserted residues, one deleted residue and 12 point substitutions. The gene was subcloned and the recombinant protein overexpressed, purified and crystallized in a trigonal space group. The structure was determined by molecular replacement and was refined to 2.1 A resolution.

A novel endonuclease mechanism directly visualized for I-PpoI.

A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.

1,3-Dioxane-4,6-dione-5-carboxamide-based inhibitors of human group IIA phospholipase A: X-ray structure of the complex and interfacial selection of inhibitors from a structural library.

A library of 109 1,3-dioxane-4,6-dione-5-carboxamides was prepared by solution-phase methods as potential inhibitors of human group IIa phospholipase A2. Tight binding inhibitors were found by an interfacial affinity selection method. The crystal structure of the secreted phospholipase A2 containing one of the inhibitors was determined, and it reveals the inhibitor-calcium bidendate coordination.

DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI.

Homing endonucleases are a diverse collection of proteins that are encoded by genes with mobile, self-splicing introns. They have also been identified in self-splicing inteins (protein introns). These enzymes promote the movement of the DNA sequences that encode them from one chromosome location to another; they do this by making a site-specific double-strand break at a target site in an allele that lacks the corresponding mobile intron. The target sites recognized by these small endonucleases are generally long (14-44 base pairs). Four families of homing endonucleases have been identified, including the LAGLIDADG, the His-Cys box, the GIY-YIG and the H-N-H endonucleases. The first identified His-Cys box homing endonuclease was I-PpoI from the slime mould Physarum polycephalum. Its gene resides in one of only a few nuclear introns known to exhibit genetic mobility. Here we report the structure of the I-PpoI homing endonuclease bound to homing-site DNA determined to 1.8 A resolution. I-PpoI displays an elongated fold of dimensions 25 x 35 x 80 A, with mixed alpha/beta topology. Each I-PpoI monomer contains three antiparallel beta-sheets flanked by two long alpha-helices and a long carboxy-terminal tail, and is stabilized by two bound zinc ions 15 A apart. The enzyme possesses a new zinc-bound fold and endonuclease active site. The structure has been determined in both uncleaved substrate and cleaved product complexes.

Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum.

The homing endonuclease I-PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26S rRNA gene. This endonuclease promotes the lateral transfer or "homing" of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26S rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I-PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombinant I-PpoI has been co-crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3(1)21, with unit cell dimensions a = b = 114 A, c = 89 A. The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A3/dalton. These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.

Calcineurin-immunosuppressor complexes.

Crystal structures of the Ser/Thr phosphatase calcineurin (protein phosphatase 2B) have recently been solved by X-ray crystallography, both in the free-protein state, and complexed with the immunophilin/immunosuppressant FKBP12/FK506. Core elements of the calcineurin phosphatase have been found to be similar to the corresponding elements of Ser/Thr phosphatase 1 and purple acid phosphatase. The structures provide a basis for understanding calcineurin inhibition by a ternary complex of immunophilin and immunosuppressant proteins.

Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion.

The sequences of two previously known tail genes, R and S, of the temperate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located between S and V, were determined. Amber mutations mapping within R and S, Ram3, Ram42, Ram23, Sam75, and Sam89 were sequenced and found to be within their corresponding open reading frames. We constructed overproducing plasmids for R and S and identified these proteins by SDS-PAGE of whole-cell lysates and Coomassie blue staining. The predicted molecular masses of proteins R and S were M(r) 17,400 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequence data. orf-30 occupies the strand opposite from RS and V and is preceded by several weak potential sigma 70-RNA polymerase promoters, some of which overlap with the V promoter. A construct that had the putative orf-30 promoter region upstream of the lacZ gene produced low levels of beta-galactosidase activity in vivo. Expression from the orf-30 promoter was not stimulated by the phage P4 transcriptional activator protein, delta, which acts at all the known P2 and P4 late promoters. Insertion mutagenesis showed that orf-30 was not an essential gene for P2 growth in Escherichia coli. None of the gene or protein sequences exhibited extensive homology to sequences in the nucleic acid and protein databases. However, the R protein contains a small region homologous to one in the phage T4 tail protein gp15, which is required for T4 tails to bind heads. We propose that R and S are tail completion proteins that are essential for stable head joining.

Yeast heat shock transcription factor contains a flexible linker between the DNA-binding and trimerization domains. Implications for DNA binding by trimeric proteins.

All heat shock transcription factors (HSFs) share two regions of homology, identified as the DNA binding and trimerization regions. The DNA binding region consists of two parts, an 89-amino-acid minimal DNA-binding domain and an additional 21 amino acids which are not necessary for specific DNA binding of a monomeric DNA-binding domain. These 21 amino acids may act as a flexible linker between the DNA-binding and trimerization domains. Saccharomyces cerevisiae HSF has an additional 52 amino acids between the proposed flexible linker and the trimerization domain. Deletion of this unique region has no effect on the structural integrity or essential in vivo functions of HSF. To investigate the role of the 21-amino-acid proposed linker, a series of internal deletions was created in fragments containing the DNA-binding and trimerization domains. The deletions have no effect on the structural integrity of the protein as assayed by circular dichroism spectroscopy. However, alterations of the linker do affect affinity of trimeric HSF binding to its target DNA. In addition, deletion of part or all of the proposed linker from full-length yeast HSF, an essential protein, disrupts growth of yeast.