PDBe: Protein Data Bank in Europe.

The Protein Data Bank in Europe (PDBe; pdbe.org) is a partner in the Worldwide PDB organization (wwPDB; wwpdb.org) and as such actively involved in managing the single global archive of biomacromolecular structure data, the PDB. In addition, PDBe develops tools, services and resources to make structure-related data more accessible to the biomedical community. Here we describe recently developed, extended or improved services, including an animated structure-presentation widget (PDBportfolio), a widget to graphically display the coverage of any UniProt sequence in the PDB (UniPDB), chemistry- and taxonomy-based PDB-archive browsers (PDBeXplore), and a tool for interactive visualization of NMR structures, corresponding experimental data as well as validation and analysis results (Vivaldi).

PDBe: Protein Data Bank in Europe.

The Protein Data Bank in Europe (PDBe) (http://www.ebi.ac.uk/pdbe/) is actively working with its Worldwide Protein Data Bank partners to enhance the quality and consistency of the international archive of bio-macromolecular structure data, the Protein Data Bank (PDB). PDBe also works closely with its collaborators at the European Bioinformatics Institute and the scientific community around the world to enhance its databases and services by adding curated and actively maintained derived data to the existing structural data in the PDB. We have developed a new database infrastructure based on the remediated PDB archive data and a specially designed database for storing information on interactions between proteins and bound molecules. The group has developed new services that allow users to carry out simple textual queries or more complex 3D structure-based queries. The newly designed 'PDBeView Atlas pages' provide an overview of an individual PDB entry in a user-friendly layout and serve as a starting point to further explore the information available in the PDBe database. PDBe's active involvement with the X-ray crystallography, Nuclear Magnetic Resonance spectroscopy and cryo-Electron Microscopy communities have resulted in improved tools for structure deposition and analysis.

Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG.

BACKGROUND: Streptococcal protein G comprises two or three domains that bind to the constant Fc region of most mammalian immunoglobulin Gs (IgGs). Protein G is functionally related to staphylococcal protein A, with which it shares neither sequence nor structural homology. RESULTS: To understand the competitive binding of these two proteins to the Fc region, the crystal structure of a single Ig-binding domain of streptococcal protein G was determined at 3.5 A resolution in complex with the Fc fragment of human IgG and compared with the structures of protein A:Fc and protein G:Fab complexes. Protein G binds to the interface between the second and third heavy chain constant domains of Fc, which is roughly the same binding site used by protein A. Protein G comprises one alpha-helix packed onto a four-stranded beta-sheet. Residues from protein G that are involved in binding are situated within the C-terminal part of the alpha-helix, the N-terminal part of the third beta-strand and the loop region connecting these two structural elements. The identified Fc-binding region of protein G agrees well with both biochemical and NMR spectroscopic data. However, the Fc-binding helices of protein G and protein A are not superimposable. CONCLUSIONS: Protein G and protein A have developed different strategies for binding to Fc. The protein G:Fc complex involves mainly charged and polar contacts, whereas protein A and Fc are held together through non-specific hydrophobic interactions and a few polar interactions. Several residues of Fc are involved in both the protein G:Fc and the protein A:Fc interaction, which explains the competitive binding of the two proteins. The apparent differences in their Fc-binding activities result from additional unique interactions.

Crystal structures of cellular retinoic acid binding proteins I and II in complex with all-trans-retinoic acid and a synthetic retinoid.

BACKGROUND: Retinoic acid (RA) plays a fundamental role in diverse cellular activities. Cellular RA binding proteins (CRABPs) are thought to act by modulating the amount of RA available to nuclear RA receptors. CRABPs and cellular retinol-binding proteins (CRBPs) share a unique fold of two orthogonal beta-sheets that encapsulate their ligands. It has been suggested that a trio of residues are the prime determinants defining the high specificity of CRBPs and CRABPs for their physiological ligands. RESULTS: Bovine/murine CRABP I and human CRABP II have been crystallized in complex with their natural ligand, all-trans-RA. Human CRABP II has also been crystallized in complex with a synthetic retinoid, 'compound 19'. Their structures have been determined and refined at resolutions of 2.9 A, 1.8 A and 2.2 A, respectively. CONCLUSIONS: The retinoid-binding site in CRABPs differs significantly from that observed in CRBP. Structural changes in three juxtaposed areas of the protein create a new, displaced binding site for RA. The carboxylate of the ligand interacts with the expected trio of residues (Arg132, Tyr134 and Arg111; CRABP II numbering). The RA ligand is almost flat with the beta-ionone ring showing a significant deviation (-33 degrees) from a cis conformation relative to the isoprene tail. The edge atoms of the beta-ionone ring are accessible to solvent in a suitable orientation for presentation to metabolizing enzymes. The bulkier synthetic retinoid causes small conformational changes in the protein structure.

Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target.

BACKGROUND: Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target. RESULTS: The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 A resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (approximately 2000 A2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme. CONCLUSIONS: Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

Validation of protein models from Calpha coordinates alone.

The Protein Data Bank contains a number of structures for which only the coordinates of the Calpha atoms have been deposited. Although many tools are available for the validation of all-atom protein models, hardly any of these can be used to assess the quality of models for which only Calpha coordinates are available. Two rapid and simple tests to assess the quality of the Calpha backbone of a protein model are described, one based on the distribution of Calpha-Calpha distances, and the other on the two-dimensional distribution of the angles and dihedrals formed by sequential Calpha atoms. Expected distributions were derived by analysing a set of 1343 high-resolution, all-atom protein models. The distance criterion is useful to discriminate between refined and unrefined models, whereas the angle/dihedral criterion can be used to discriminate between normal and possibly problematic Calpha models. The method has been applied to a set of 88 Calpha-only models from the Protein Data Bank. The tracing of two of the models that are outliers in this analysis has recently been shown to be incorrect. Other applications of the method are discussed.

Recognition of spatial motifs in protein structures.

As the structural database continues to expand, new methods are required to analyse and compare protein structures. Whereas the recognition, comparison, and classification of folds is now more or less a solved problem, tools for the study of constellations of small numbers of residues are few and far between. In this paper, two programs are described for the analysis of spatial motifs in protein structures. The first, SPASM, can be used to find the occurrence of a motif consisting of arbitrary main-chain and/or side-chains in a database of protein structures. The program also has a unique capability to carry out "fuzzy pattern matching" with relaxed requirements on the types of some or all of the matching residues. The second program, RIGOR, scans a single protein structure for the occurrence of any of a set of pre-defined motifs from a database. In one application, spatial motif recognition combined with profile analysis enabled the assignment of the structural and functional class of an uncharacterised hypothetical protein in the sequence database. In another application, the occurrence of short left-handed helical segments in protein structures was investigated, and such segments were found to be fairly common. Potential applications of the techniques presented here lie in the analysis of (newly determined) structures, in comparative structural analysis, in the design and engineering of novel functional sites, and in the prediction of structure and function of uncharacterised proteins.

The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 A resolution, and a comparison with related enzymes.

Cellulose is the most abundant polymer in the biosphere. Although generally resistant to degradation, it may be hydrolysed by cellulolytic organisms that have evolved a variety of structurally distinct enzymes, cellobiohydrolases and endoglucanases, for this purpose. Endoglucanase I (EG I) is the major endoglucanase produced by the cellulolytic fungus Trichoderma reesei, accounting for 5 to 10% of the total amount of cellulases produced by this organism. Together with EG I from Humicola insolens and T. reesei cellobiohydrolase I (CBH I), the enzyme is classified into family 7 of the glycosyl hydrolases, and it catalyses hydrolysis with a net retention of the anomeric configuration. The structure of the catalytic core domain (residues 1 to 371) of EG I from T. reesei has been determined at 3.6 A resolution by the molecular replacement method using the structures of T. reesei CBH I and H. insolens EG I as search models. By employing the 2-fold non-crystallographic symmetry (NCS), the structure was refined successfully, despite the limited resolution. The final model has an R-factor of 0.201 (Rfree 0.258). The structure of EG I reveals an extended, open substrate-binding cleft, rather than a tunnel as found in the homologous cellobiohydrolase CBH I. This confirms the earlier proposal that the tunnel-forming loops in CBH I have been deleted in EG I, which has resulted in an open active site in EG I, enabling it to function as an endoglucanase. Comparison of the structure of EG I with several related enzymes reveals structural similarities, and differences that relate to their biological function in degrading particular substrates. A possible structural explanation of the drastically different pH profiles of T. reesei and H. insolens EG I is proposed.

Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the Mu and Pi class enzymes.

The crystal structure of human alpha class glutathione transferase A1-1 has been determined and refined to a resolution of 2.6 A. There are two copies of the dimeric enzyme in the asymmetric unit. Each monomer is built from two domains. A bound inhibitor, S-benzyl-glutathione, is primarily associated with one of these domains via a network of hydrogen bonds and salt-links. In particular, the sulphur atom of the inhibitor forms a hydrogen bond to the hydroxyl group of Tyr9 and the guanido group of Arg15. The benzyl group of the inhibitor is completely buried in a hydrophobic pocket. The structure shows an overall similarity to the mu and pi class enzymes particularly in the glutathione-binding domain". The main difference concerns the extended C terminus of the alpha class enzyme which forms an extra alpha-helix that blocks one entrance to the active site and makes up part of the substrate binding site.

Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein.

Acyl-CoA binding protein (ACBP) maintains a pool of fatty acyl-CoA molecules in the cell and plays a role in fatty acid metabolism. The biochemical properties of Plasmodium falciparum ACBP are described together with the 2.0 A resolution crystal structures of a P. falciparum ACBP-acyl-CoA complex and of bovine ACBP in two crystal forms. Overall, the bovine ACBP crystal structures are similar to the NMR structures published previously; however, the bovine and parasite ACBP structures are less similar. The parasite ACBP is shown to have a different ligand-binding pocket, leading to an acyl-CoA binding specificity different from that of bovine ACBP. Several non-conservative differences in residues that interact with the ligand were identified between the mammalian and parasite ACBPs. These, together with measured binding-specificity differences, suggest that there is a potential for the design of molecules that might selectively block the acyl-CoA binding site.

Use of non-crystallographic symmetry in protein structure refinement.

Several methods to assess the (dis)similarity of protein structures objectively are described, some of which, when applied to non-crystallographically related protein models, are able to discriminate between significant differences and 'random noise'. Some of these methods have been used to investigate a sample of several hundred protein structures which have been solved by means of X-ray crystallography in order to investigate the extent to which non-crystallographically related protein models differ from one another. It is shown that the extent of such differences is largely dependent on the resolution of the data used for the determination and refinement of the structure and, measured by some statistics, even varies essentially linearly with the resolution. The implications of these findings for the strategies used to refine structures with non-crystallographic symmetry, in particular at low resolution, are discussed. Finally, two examples are given of recent structure determinations from this laboratory in which the presence (and employment) of non-crystallographic symmetry was crucial to the solution and refinement of the structure.

Experimental assessment of differences between related protein crystal structures.

Prior to attaching any biological significance to differences between two related protein crystal structures, it must be established that such differences are genuine, rather than artefacts of the structure-determination protocol. This will be all the more important as more and more related protein structures are solved and comparative structural biology attempts to correlate structural differences with variations in biological function, activity or affinity. A method has been developed which enables unbiased assessment of differences between the structures of related biomacromolecules using experimental crystallographic information alone. It is based on the use of local density-correlation maps, which contain information regarding the similarity of the experimental electron density for corresponding parts of different copies of a molecule. The method can be used to assess a priori which parts of two or more molecules are likely to be structurally similar; this information can then be employed during structure refinement. Alternatively, the method can be used a posteriori to verify that differences observed in two or more models are supported by the experimental information. Several examples are discussed which validate the notion that local conformational variability is highly correlated to differences in the local experimental electron density.