Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 Å resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium

Crystallization of YIoQ, a GTPase of unknown function essential for Bacillus subtilis viability

YLoQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 Angstrom spacing

Crystallization of the oligopeptide-binding protein AppA from Bacillus subtilis

AppA is the membrane-anchored extracellular receptor component of an ABC transporter responsible for the uptake of oligopeptides into Bacillus subtilis. AppA has been overexpressed as a cleavable maltose-binding protein fusion in Escherichia coli. Following removal of the fusion portion, AppA has been crystallized from morpholino-ethanesulfonic acid-buffered solutions at pH 6.5 containing polyethylene glycol and zinc acetate. A complete X-ray diffraction data set extending to 2.3 Angstrom spacing has been collected

The structure of Rph, an exoribonuclease from Bacillus anthracis, at 1.7 angstrom resolution

Maturation of tRNA precursors into functional tRNA molecules requires trimming of the primary transcript at both the 5' and 3' ends. Cleavage of nucleotides from the 3' stem of tRNA precursors, releasing nucleotide diphosphates, is accomplished in Bacillus by a phosphate-dependent exoribonuclease, Rph. The crystal structure of this enzyme from B. anthracis has been solved by molecular replacement to a resolution of 1.7 angstrom and refined to an R factor of 19.3%. There is one molecule in the asymmetric unit; the crystal packing reveals the assembly of the protein into a hexamer arranged as a trimer of dimers. The structure shows two sulfate ions bound in the active-site pocket, probably mimicking the phosphate substrate and the phosphate of the 3'-terminal nucleotide of the tRNA precursor. Three other bound sulfate ions point to likely RNA-binding sites

Structure of the Branched Chain Amino Acid and GTP Sensing Global Regulator, CodY, from Bacillus subtilis

CodY is a branched-chain amino acid (BCAA) and GTP sensor, and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. CodY consists of a GAF domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA-dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn alpha-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganised GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36 base-pair operator fragment are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed

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 p/ 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 Km for the hydrolysis of the latter substrate is 6 mM. The enzyme activity is optimal at 55°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 × 0.15 × 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 Å; b = 61.61 Å; c = 56.11 Å; γ = 117.57°. The X-ray data set to 1.7 Å resolution has been collected on an automatic synchrotron (EMBL Hamburg Station)

Human Lin28 forms a high-affinity 1:1 complex with the 106~363 cluster miRNA miR-363

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumour suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity toward this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterisation of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with 1:1 stoichiometry and with similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model where the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions

Knotted vs. Unknotted Proteins: Evidence of Knot-Promoting Loops

Knotted proteins, because of their ability to fold reversibly in the same topologically entangled conformation, are the object of an increasing number of experimental and theoretical studies. The aim of the present investigation is to assess, on the basis of presently available structural data, the extent to which knotted proteins are isolated instances in sequence or structure space, and to use comparative schemes to understand whether specific protein segments can be associated to the occurrence of a knot in the native state. A significant sequence homology is found among a sizeable group of knotted and unknotted proteins. In this family, knotted members occupy a primary sub-branch of the phylogenetic tree and differ from unknotted ones only by additional loop segments. These "knot-promoting" loops, whose virtual bridging eliminates the knot, are found in various types of knotted proteins. Valuable insight into how knots form, or are encoded, in proteins could be obtained by targeting these regions in future computational studies or excision experiments