1.2 Angstroms crystal structure of the S. pneumoniae PhtA histidine triad domain a novel zinc binding fold.

The recently described pneumococcal histidine triad protein family has been shown to be highly conserved within the pneumococcus. As part of our structural genomics effort on proteins from Streptococcus pneumoniae, we have expressed, crystallised and solved the structure of PhtA-166-220 at 1.2 Angstroms using remote SAD with zinc. The structure of PhtA-166-220 shows no similarity to any protein structure. The overall fold contains 3beta-strands and a single short alpha-helix. The structure appears to contain a novel zinc binding motif. The remaining 4 histidine triad repeats from PhtA have been modelled based on the crystal structure of the PhtA histidine triad repeat 2. From this modelling work, we speculate that only three of the five histidine triad repeats contain the residues in the correct geometry to allow the binding of a zinc ion.

Cytokine gene polymorphisms in idiopathic pulmonary fibrosis.

Pro- and anti-fibrotic cytokine gene polymorphisms may affect expression of idiopathic pulmonary fibrosis (IPF). The aims of the present case-control study were to examine polymorphisms in the IL-6, transforming growth factor (TGF)-beta 1, tumour necrosis factor (TNF)-alpha and interleukin-1 (IL-1)Ra genes in patients with IPF (n = 22) -compared to healthy controls (n = 140). Genotyping was performed on DNA extracted from peripheral blood lymphocytes, using polymerase chain reaction - restriction fragment length polymorphism with gene polymorphisms determined according to -published techniques. The following sites were examined: (i) IL-1Ra*1-5 (86 bp variable tandem repeat intron 2), (ii) IL-6 (-174G > C), (iii) TNF-alpha (-308G > A) and (iv) TGF-beta 1 (Arg25Pro). The TNF-alpha (-308 A) allele was over-represented in the IPF (p(corr) = 0.004) group compared to controls. Risk of IPF was significant for heterozygotes for: (i) the TNF-alpha (-308 A) allele (A/G) (odds ratio (OR) 2.9; 95% confidence interval (CI) 1.2-7.2; P = 0.02), (ii) homozygotes (A/A) (OR 13.9; 95%CI 1.2-160; P = 0.04) and (iii) carriage of the allele (A/A + A/G) (OR 4; 95%CI 1.6-10.2; P = 0.003). The distribution of alleles and genotypes for IL-6, TGF-beta 1 and IL-1Ra between the two groups was not significantly different. This is the third study to independently confirm that there is a significant association of the TNF-alpha (-308 A) allele with IPF. Further research is needed to assess the utility of cytokine gene polymorphisms as markers of disease -susceptibility.

Mannose-binding lectin gene polymorphism predicts hospital admissions for COPD infections.

Infection frequently causes exacerbations of chronic obstructive pulmonary disease (COPD). Mannose-binding lectin (MBL) is a pattern-recognition receptor that assists in clearing microorganisms. Polymorphisms in the MBL2 gene reduce serum MBL levels and are associated with risk of infection. We studied whether the MBL2 codon 54 B allele affected serum MBL levels, admissions for infective exacerbation in COPD and disease susceptibility. Polymorphism frequency was determined by PCR-RFLP in 200 COPD patients and 104 smokers with normal lung function. Serum MBL was measured as mannan-binding activity in a subgroup of 82 stable COPD patients. Frequency of COPD admissions for infective exacerbation was ascertained for a 2-year period. The MBL2 codon 54 B allele reduced serum MBL in COPD patients. In keeping, patients carrying the low MBL-producing B allele had increased risk of admission for infective exacerbation (OR 4.9, P(corrected)=0.011). No association of MBL2 genotype with susceptibility to COPD was detected. In COPD, serum MBL is regulated by polymorphism at codon 54 in its encoding gene. Low MBL-producing genotypes were associated with more frequent admissions to hospital with respiratory infection, suggesting that the MBL2 gene is disease-modifying in COPD. MBL2 genotype should be explored prospectively as a prognostic marker for infection risk in COPD.

Crystal structure of Mip, a prolylisomerase from Legionella pneumophila.

The human pathogen Legionella pneumophila, the etiological agent of the severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits a peptidyl prolyl cis-trans isomerase (PPIase) activity, which appears to be important for infection. Here we report the 2.4 A crystal structure of the Mip protein from L. pneumophila Philadelphia 1 and the 3.2 A crystal structure of its complex with the drug FK506. Each monomer of the homodimeric protein consists of an N-terminal dimerization module, a long (65 A) connecting alpha-helix and a C-terminal PPIase domain exhibiting similarity to human FK506-binding protein. In view of the recent significant increase in the number of reported cases of Legionnaires' disease and other intracellular infections, these structural results are of prime interest for the design of new drugs directed against Mip proteins of intracellular pathogens.

Crystal structure of the anti-fungal target N-myristoyl transferase.

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel alpha/beta fold which shows internal two-fold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.

The entropic penalty of ordered water accounts for weaker binding of the antibiotic novobiocin to a resistant mutant of DNA gyrase: a thermodynamic and crystallographic study.

Novobiocin is an antibiotic which binds to a 24 kDa fragment from the B subunit of DNA gyrase. Naturally occurring resistance arises from mutation of Arg-136 which hydrogen bonds to the coumarin ring of novobiocin. We have applied calorimetry to characterize the binding of novobiocin to wild-type and R136H mutant 24 kDa fragments. Upon mutation, the Kd increases from 32 to 1200 nM at 300 K. The enthalpy of binding is more favorable for the mutant (DeltaH degrees shifts from -12.1 to -17.5 kcal/mol), and the entropy of binding is much less favorable (TDeltaS degrees changes from -1.8 to -9.4 kcal/mol). Both of these changes are in the direction opposite to that expected if the loss of the Arg residue reduces hydrogen bonding. The change in heat capacity at constant pressure upon binding (DeltaCp) shifts from -295 to -454 cal mol-1 K-1. We also report the crystal structure, at 2.3 A resolution, of a complex between the R136H 24 kDa fragment and novobiocin. Although the change in DeltaCp often would be interpreted as reflecting increased burial of hydrophobic surface on binding, this structure reveals a small decrease. Furthermore, an ordered water molecule is sequestered into the volume vacated by removal of the guanidinium group. There are large discrepancies when the measured thermodynamic parameters are compared to those estimated from the structural data using empirical relationships. These differences seem to arise from the effects of sequestering ordered water molecules upon complexation. The water-mediated hydrogen bonds linking novobiocin to the mutant protein make a favorable enthalpic contribution, whereas the immobilization of the water leads to an entropic cost and a reduction in the heat capacity of the system. Such a negative contribution to DeltaCp, DeltaH degrees , and TDeltaS degrees appears to be a general property of water molecules that are sequestered when ligands bind to proteins.

Evaluation of colostrum and whey-derived gamma globulins for prevention of cryptosporidiosis in artificially infected neonatal calves.

Thirty colostrum-deprived Friesian male calves were used to study the effect of colostrum and whey-derived gammaglobulins on the clinical events and oocyst-excretion pattern following artificial infection with a fresh, field-sourced strain of Cryptosporidium parvum. The calves were raised naive and free from contact with C. parvum from birth until infection at either 10 or 17 days of age. The two age groups comprised three sub-groups that were each given a single treatment of colostrum, whey-derived gammaglobulins or whole milk (controls) at 5 hours after birth. Blood samples taken both before and 48 hours after this dosing showed the mean serum gamma globulin concentrations changed from almost zero in all calves to 462,279 and 29 mg/dl for the colostrum, whey-derived gammaglobulin and whole milk sub-groups respectively. The results showed that a majority of calves shed Cryptosporidium oocysts within 7 days of oral infection but that no diarrhoea or other clinical signs were associated with this. However, when the level of C. parvum faecal shedding was graded, the results showed a trend towards a higher level of oocyst shedding and over a longer period of time in the control calves deprived of any passive immunity than in the two groups given either colostrum or whey-derived gammaglobulins.

The preparation and properties of immobilised dipeptidyl-aminopeptidase I (cathepsin C).

Dipeptidyl-aminopeptidase I (dipeptidyl-peptide hydrolase, EC 3.4.14.1) from bovine spleen has been immobilized by hydrophobic bonding to alkyl- or aryl-Sepharoses. Optimum binding occurred with octyl- and phenyl-Sepharoses. The activity of the immobilised dipeptidyl-aminopeptidase I has been determined using glycylarginyl-p-nitroanilide as substrate and the pH optimum of the immobilised enzyme determined as well as the stability of the enzyme to repeated use. Preliminary studies using immobilised dipeptidyl-aminopeptidase I for the digestion of methionine enkephalin have been carried out using reverse-phase HPLC to analyse the reaction.