Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis.

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

Atomic structure of the Serratia marcescens endonuclease at 1.1 A resolution and the enzyme reaction mechanism.

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.

BACKGROUND: The biosynthesis of key metabolic components is of major interest to biologists. Studies of de novo purine synthesis are aimed at obtaining a deeper understanding of this central pathway and the development of effective chemotherapeutic agents. Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase catalyses the seventh step out of ten in the biosynthesis of purine nucleotides. To date, only one structure of an enzyme involved in purine biosynthesis has been reported: adenylosuccinate synthetase, which catalyses the first committed step in the synthesis of AMP from IMP. RESULTS: We report the first three-dimensional structure of a SAICAR synthase, from Saccharomyces cerevisiae. It is a monomer with three domains. The first two domains consist of antiparallel beta sheets and the third is composed of two alpha helices. There is a long deep cleft made up of residues from all three domains. Comparison of SAICAR synthases by alignment of their sequences reveals a number of conserved residues, mostly located in the cleft. The presence of two sulphate ions bound in the cleft, the structure of SAICAR synthase in complex with ATP and a comparison of this structure with that of other ATP-dependent proteins point to the interdomain cleft as the location of the active site. CONCLUSIONS: The topology of the first domain of SAICAR synthase resembles that of the N-terminal domain of proteins belonging to the cyclic AMP-dependent protein kinase family. The fold of the second domain is similar to that of members of the D-alanine:D-alanine ligase family. Together these enzymes form a new superfamily of mononucleotide-binding domains. There appears to be no other enzyme, however, which is composed of the same combination of three domains, with the individual topologies found in SAICAR synthase.

Glutamyl endopeptidase of Bacillus intermedius strain 3-19. Purification, properties, and crystallization.

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic and, rarely, of aspartic acid residues in peptides and proteins was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM-cellulose and Mono S. The enzyme molecular mass is 29 kD and the pI is 8.4. The proteinase is inhibited by DFP. The enzyme, like other glutamyl endopeptidases, reveals two pH optima (pH 7.5 and 9.0) for casein and one (pH 8.0) for Z-Glu-pNA hydrolysis. The K(m) for the hydrolysis of the latter substrate is 6 mM. The enzyme activity is optimal at 55 degrees C. The enzyme is stable in the pH range 6.5-11.0. Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase. Crystal prisms or plates 0.25-0.3 x 0.15 x 0.07-0.1 mm have been grown using the vapor diffusion technique in a hanging drop followed by macroseeding. The crystals belong to the space group B2 with the following unit cell parameters: a = 69.59 A; b = 61.61 A; c = 56.11 A; gamma = 117.57 degrees. The X-ray data set to 1.7 A resolution has been collected on an automatic synchrotron (EMBL Hamburg Station).

Three-dimensional structure of Serratia marcescens nuclease at 1.7 A resolution and mechanism of its action.

The three-dimensional crystal structure of Serratia marcescens (Sm) nuclease has been refined at 1.7 A resolution to the R-factor of 17.3% and R-free of 22.2%. The final model consists of 3678 non-hydrogen atoms and 443 water molecules. The analysis of the secondary and the tertiary structures of the Sm nuclease suggests a topology which reveals essential inner symmetry in all the three layers forming the monomer. We propose the plausible mechanism of its action based on a concerted participation of the catalytically important amino acid residues of the enzyme active site.

Crystallization and preliminary X-ray investigation of phosphoribosylaminoimidazolesuccinocarboxamide synthase from the yeast Saccharomyces cerevisiae.

Crystals of phosphoribosylaminoimidazolesuccinocarboxamide synthase (EC 6.3.2.6) from the yeast Saccharomyces cerevisiae were grown by the vapor diffusion hanging-drop technique, using ammonium sulfate as the precipitant. The crystals had dimensions up to 1.2 mm. X-ray diffraction experiments indicated a space group of P2(1)2(1)2(1) and unit cell parameters of a = 62.3 A, b = 63.5 A and c = 80.9 A, with one molecule in the asymmetric unit. Native data have been collected to 2.5 A resolution.