Inference of macromolecular assemblies from crystalline state

We discuss basic physical-chemical principles underlying the formation of stable macromolecular complexes, which in many cases are likely to be the biological units performing a certain physiological function. We also consider available theoretical approaches to the calculation of macromolecular affinity and entropy of complexation. The latter is shown to play an important role and make a major effect on complex size and symmetry. We develop a new method, based on chemical thermodynamics, for automatic detection of macromolecular assemblies in the Protein Data Bank (PDB) entries that are the results of X-ray diffraction experiments. As found, biological units may be recovered at 80-90% success rate, which makes X-ray crystallography an important source of experimental data on macromolecular complexes and protein-protein interactions. The method is implemented as a public WWW service.     

Expert opinion as 'validation' of risk assessment applied to calf welfare

Background  

Recently, a Risk Assessment methodology was applied to animal welfare issues in a report of the European Food Safety Authority (EFSA) on intensively housed calves.     

BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions

We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems.     

Molecular evolution of a Y-chromosomal repetitive sequence family in the genus Mus

A 522-base-long Y-chromosomal sequence was isolated from a BALB/c genomic library and was designated "BF046." It is repeated about 200 times in the male genome, and a difference was detected between the Mus musculus musculus and the M. m. domesticus type Y chromosomes. BF046- related sequences were present over the entire length of the Y chromosome as visualized by in situ hybridization. Southern blot analysis against DNAs isolated from eight species in the genus Mus showed that BF046-related sequences were amplified in the Y chromosomes of three closely related species: M. musculus, M. spicilegus, and M. spretus. To gain insight into the stability of the BF046 sequence family, we isolated 18 additional clones from these three mouse species and compared their sequences. The M. musculus sequences differed from the M. spicilegus and M. spretus sequences by two indels. The remaining parts of the sequences were very similar, but both parsimony and distance-based analytical methods divided the sequences into the same four subgroups, with each species having its own subgroup(s). Thus, the Y chromosomes of M. musculus, M. spicilegus, and M. spretus can be distinguished from one another.      

Assessment of changes in the brca2 and p53 genes in breast invasive ductal carcinoma in northeast Brazil

Background

BRCA protein interacts with at least 13 different proteins that have been implicated with cancer susceptibility and loss of BRCA function is correlated to sensitivity to DNA crosslinking agents in preclinical models.

Results

BRCA2 methylation frequency was 44%, p53 Pro22 allele frequency was 32% and heterozygous frequency of Arg/Pro72 genotype was 60% which could be associated as risk factor for metastasis (p = 0.046 OR = 4.190). Regarding to polymorphism of codon 249 the frequency of Arg249 allele presented 82% which was considered not statistically significant.

Conclusions

There was not statistical significance to BRCA2 promoter methylation with any parameters chosen. However, our findings suggest that patients who present heterozygous genotype at codon 72 of p53 gene may have a major susceptibility to any type of metastasis and this could serve as potential auxiliary biomarker for poor prognosis.     

The R-spondin protein family

The four vertebrate R-spondin proteins are secreted agonists of the canonical Wnt/β-catenin signaling pathway. These proteins are approximately 35 kDa, and are characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal potentiation, and a thrombospondin domain situated more towards the carboxyl terminus that can bind matrix glycosaminoglycans and/or proteoglycans. Although R-spondins are unable to initiate Wnt signaling, they can potently enhance responses to low-dose Wnt proteins. In humans, rare disruptions of the gene encoding R-spondin1 cause a syndrome of XX sex reversal (phenotypic male), palmoplantar keratosis (a thickening of the palms and soles caused by excess keratin formation) and predisposition to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 cause anonychia (absence or hypoplasia of nails on fingers and toes). Recently, leucine-rich repeat-containing G-protein-coupled receptor (Lgr)4, Lgr5 and Lgr6, three closely related orphans of the leucine-rich repeat family of G-protein-coupled receptors, have been identified as receptors for R-spondins. Lgr5 and Lgr6 are markers for adult stem cells. Because R-spondins are potent stimulators of adult stem cell proliferation in vivo and in vitro, these findings might guide the therapeutic use of R-spondins in regenerative medicine.     

Pound-wise but penny-foolish: How well do micromolecules fare in macromolecular refinement?

For the refinement of protein and nucleic acid structures, high-quality geometric restraint libraries are available. Unfortunately, for other compounds, such as physiological ligands, lead compounds, substrate analogs, etc., the situation is not as favorable. As a result, the structures of small molecules found in complexes with biomacromolecules are often less reliable than those of the surrounding amino or nucleic acids. Here, we briefly review the use of geometric restraints in structure refinement (be it against X-ray crystallographic or NMR-derived data) and simulation. In addition, we discuss methods to generate both restraint libraries and (idealized) coordinates for small molecules and provide some practical advice.     

The European Bioinformatics Institute's data resources

As the amount of biological data grows, so does the need for biologists to store and access this information in central repositories in a free and unambiguous manner. The European Bioinformatics Institute (EBI) hosts six core databases, which store information on DNA sequences (EMBL-Bank), protein sequences (SWISS-PROT and TrEMBL), protein structure (MSD), whole genomes (Ensembl) and gene expression (ArrayExpress). But just as a cell would be useless if it couldn't transcribe DNA or translate RNA, our resources would be compromised if each existed in isolation. We have therefore developed a range of tools that not only facilitate the deposition and retrieval of biological information, but also allow users to carry out searches that reflect the interconnectedness of biological information. The EBI's databases and tools are all available on our website at www.ebi.ac.uk.     

Discriminating between homodimeric and monomeric proteins in the crystalline state

Scores calculated from intermolecular contacts of proteins in the crystalline state are used to differentiate monomeric and homodimeric proteins, by classification into two categories separated by a cut-off score value. The generalized classification error is estimated by using bootstrap re-sampling on a nonredundant set of 172 water-soluble proteins whose prevalent quaternary state in solution is known to be either monomeric or homodimeric. A statistical potential, based on atom-pair frequencies across interfaces observed with homodimers, is found to yield an error rate of 12.5%. This indicates a small but significant improvement over the measure of solvent accessible surface area buried in the contact interface, which achieves an error rate of 15.4%. A further modification of the latter parameter relating the two most extensive contacts of the crystal results in an even lower error rate of 11.1%.     

The lactosylceramide binding specificity of Helicobacter pylori

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.