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OneDep, a unified system for deposition, biocuration, and validation of experimentally determined structures of biological macromolecules to the PDB archive, has been developed as a global collaboration by the worldwide PDB (wwPDB) partners. This new system was designed to ensure that the wwPDB could meet the evolving archiving requirements of the scientific community over the coming decades. OneDep unifies deposition, biocuration, and validation pipelines across all wwPDB, EMDB, and BMRB deposition sites with improved focus on data quality and completeness in these archives, while supporting growth in the number of depositions and increases in their average size and complexity. In this paper, we describe the design, functional operation, and supporting infrastructure of the OneDep system, and provide initial performance assessments., (Published by Elsevier Ltd.)

Congenital heart defects (CHDs) have a neonatal incidence of 0.8-1% (refs. 1,2). Despite abundant examples of monogenic CHD in humans and mice, CHD has a low absolute sibling recurrence risk (∼2.7%), suggesting a considerable role for de novo mutations (DNMs) and/or incomplete penetrance. De novo protein-truncating variants (PTVs) have been shown to be enriched among the 10% of 'syndromic' patients with extra-cardiac manifestations. We exome sequenced 1,891 probands, including both syndromic CHD (S-CHD, n = 610) and nonsyndromic CHD (NS-CHD, n = 1,281). In S-CHD, we confirmed a significant enrichment of de novo PTVs but not inherited PTVs in known CHD-associated genes, consistent with recent findings. Conversely, in NS-CHD we observed significant enrichment of PTVs inherited from unaffected parents in CHD-associated genes. We identified three genome-wide significant S-CHD disorders caused by DNMs in CHD4, CDK13 and PRKD1. Our study finds evidence for distinct genetic architectures underlying the low sibling recurrence risk in S-CHD and NS-CHD.

Discovery of most autosomal recessive disease-associated genes has involved analysis of large, often consanguineous multiplex families or small cohorts of unrelated individuals with a well-defined clinical condition. Discovery of new dominant causes of rare, genetically heterogeneous developmental disorders has been revolutionized by exome analysis of large cohorts of phenotypically diverse parent-offspring trios. Here we analyzed 4,125 families with diverse, rare and genetically heterogeneous developmental disorders and identified four new autosomal recessive disorders. These four disorders were identified by integrating Mendelian filtering (selecting probands with rare, biallelic and putatively damaging variants in the same gene) with statistical assessments of (i) the likelihood of sampling the observed genotypes from the general population and (ii) the phenotypic similarity of patients with recessive variants in the same candidate gene. This new paradigm promises to catalyze the discovery of novel recessive disorders, especially those with less consistent or nonspecific clinical presentations and those caused predominantly by compound heterozygous genotypes.

There are few better examples of the need for data sharing than in the rare disease community, where patients, physicians, and researchers must search for "the needle in a haystack" to uncover rare, novel causes of disease within the genome. Impeding the pace of discovery has been the existence of many small siloed datasets within individual research or clinical laboratory databases and/or disease-specific organizations, hoping for serendipitous occasions when two distant investigators happen to learn they have a rare phenotype in common and can "match" these cases to build evidence for causality. However, serendipity has never proven to be a reliable or scalable approach in science. As such, the Matchmaker Exchange (MME) was launched to provide a robust and systematic approach to rare disease gene discovery through the creation of a federated network connecting databases of genotypes and rare phenotypes using a common application programming interface (API). The core building blocks of the MME have been defined and assembled. Three MME services have now been connected through the API and are available for community use. Additional databases that support internal matching are anticipated to join the MME network as it continues to grow., (© 2015 WILEY PERIODICALS, INC.)

DECIPHER (https://decipher.sanger.ac.uk) is a web-based platform for secure deposition, analysis, and sharing of plausibly pathogenic genomic variants from well-phenotyped patients suffering from genetic disorders. DECIPHER aids clinical interpretation of these rare sequence and copy-number variants by providing tools for variant analysis and identification of other patients exhibiting similar genotype-phenotype characteristics. DECIPHER also provides mechanisms to encourage collaboration among a global community of clinical centers and researchers, as well as exchange of information between clinicians and researchers within a consortium, to accelerate discovery and diagnosis. DECIPHER has contributed to matchmaking efforts by enabling the global clinical genetics community to identify many previously undiagnosed syndromes and new disease genes, and has facilitated the publication of over 700 peer-reviewed scientific publications since 2004. At the time of writing, DECIPHER contains anonymized data from ∼250 registered centers on more than 51,500 patients (∼18000 patients with consent for data sharing and ∼25000 anonymized records shared privately). In this paper, we describe salient features of the platform, with special emphasis on the tools and processes that aid interpretation, sharing, and effective matchmaking with other data held in the database and that make DECIPHER an invaluable clinical and research resource., (© 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.)

Background: Human genome sequencing has transformed our understanding of genomic variation and its relevance to health and disease, and is now starting to enter clinical practice for the diagnosis of rare diseases. The question of whether and how some categories of genomic findings should be shared with individual research participants is currently a topic of international debate, and development of robust analytical workflows to identify and communicate clinically relevant variants is paramount., Methods: The Deciphering Developmental Disorders (DDD) study has developed a UK-wide patient recruitment network involving over 180 clinicians across all 24 regional genetics services, and has performed genome-wide microarray and whole exome sequencing on children with undiagnosed developmental disorders and their parents. After data analysis, pertinent genomic variants were returned to individual research participants via their local clinical genetics team., Findings: Around 80,000 genomic variants were identified from exome sequencing and microarray analysis in each individual, of which on average 400 were rare and predicted to be protein altering. By focusing only on de novo and segregating variants in known developmental disorder genes, we achieved a diagnostic yield of 27% among 1133 previously investigated yet undiagnosed children with developmental disorders, whilst minimising incidental findings. In families with developmentally normal parents, whole exome sequencing of the child and both parents resulted in a 10-fold reduction in the number of potential causal variants that needed clinical evaluation compared to sequencing only the child. Most diagnostic variants identified in known genes were novel and not present in current databases of known disease variation., Interpretation: Implementation of a robust translational genomics workflow is achievable within a large-scale rare disease research study to allow feedback of potentially diagnostic findings to clinicians and research participants. Systematic recording of relevant clinical data, curation of a gene-phenotype knowledge base, and development of clinical decision support software are needed in addition to automated exclusion of almost all variants, which is crucial for scalable prioritisation and review of possible diagnostic variants. However, the resource requirements of development and maintenance of a clinical reporting system within a research setting are substantial., Funding: Health Innovation Challenge Fund, a parallel funding partnership between the Wellcome Trust and the UK Department of Health., (Copyright © 2015 Wright et al. Open Access article distributed under the terms of CC BY. Published by Elsevier Ltd. All rights reserved.)

Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10(-7)) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The DECIPHER database (https://decipher.sanger.ac.uk/) is an accessible online repository of genetic variation with associated phenotypes that facilitates the identification and interpretation of pathogenic genetic variation in patients with rare disorders. Contributing to DECIPHER is an international consortium of >200 academic clinical centres of genetic medicine and ≥1600 clinical geneticists and diagnostic laboratory scientists. Information integrated from a variety of bioinformatics resources, coupled with visualization tools, provides a comprehensive set of tools to identify other patients with similar genotype-phenotype characteristics and highlights potentially pathogenic genes. In a significant development, we have extended DECIPHER from a database of just copy-number variants to allow upload, annotation and analysis of sequence variants such as single nucleotide variants (SNVs) and InDels. Other notable developments in DECIPHER include a purpose-built, customizable and interactive genome browser to aid combined visualization and interpretation of sequence and copy-number variation against informative datasets of pathogenic and population variation. We have also introduced several new features to our deposition and analysis interface. This article provides an update to the DECIPHER database, an earlier instance of which has been described elsewhere [Swaminathan et al. (2012) DECIPHER: web-based, community resource for clinical interpretation of rare variants in developmental disorders. Hum. Mol. Genet., 21, R37-R44].

Patients with developmental disorders often harbour sub-microscopic deletions or duplications that lead to a disruption of normal gene expression or perturbation in the copy number of dosage-sensitive genes. Clinical interpretation for such patients in isolation is hindered by the rarity and novelty of such disorders. The DECIPHER project (https://decipher.sanger.ac.uk) was established in 2004 as an accessible online repository of genomic and associated phenotypic data with the primary goal of aiding the clinical interpretation of rare copy-number variants (CNVs). DECIPHER integrates information from a variety of bioinformatics resources and uses visualization tools to identify potential disease genes within a CNV. A two-tier access system permits clinicians and clinical scientists to maintain confidential linked anonymous records of phenotypes and CNVs for their patients that, with informed consent, can subsequently be shared with the wider clinical genetics and research communities. Advances in next-generation sequencing technologies are making it practical and affordable to sequence the whole exome/genome of patients who display features suggestive of a genetic disorder. This approach enables the identification of smaller intragenic mutations including single-nucleotide variants that are not accessible even with high-resolution genomic array analysis. This article briefly summarizes the current status and achievements of the DECIPHER project and looks ahead to the opportunities and challenges of jointly analysing structural and sequence variation in the human genome.

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).

The Protein Data Bank in Europe (PDBe; pdbe.org) is actively involved in managing the international archive of biomacromolecular structure data as one of the partners in the Worldwide Protein Data Bank (wwPDB; wwpdb.org). PDBe also develops new tools to make structural data more widely and more easily available to the biomedical community. PDBe has developed a browser to access and analyze the structural archive using classification systems that are familiar to chemists and biologists. The PDBe web pages that describe individual PDB entries have been enhanced through the introduction of plain-English summary pages and iconic representations of the contents of an entry (PDBprints). In addition, the information available for structures determined by means of NMR spectroscopy has been expanded. Finally, the entire web site has been redesigned to make it substantially easier to use for expert and novice users alike. PDBe works closely with other teams at the European Bioinformatics Institute (EBI) and in the international scientific community to develop new resources with value-added information. The SIFTS initiative is an example of such a collaboration--it provides extensive mapping data between proteins whose structures are available from the PDB and a host of other biomedical databases. SIFTS is widely used by major bioinformatics resources.

The Protein Data Bank (PDB) is the repository for three-dimensional structures of biological macromolecules, determined by experimental methods. The data in the archive is free and easily available via the Internet from any of the worldwide centers managing this global archive. These data are used by scientists, researchers, bioinformatics specialists, educators, students, and general audiences to understand biological phenomenon at a molecular level. Analysis of this structural data also inspires and facilitates new discoveries in science. This chapter describes the tools and methods currently used for deposition, processing, and release of data in the PDB. References to future enhancements are also included.

The Protein Data Bank (PDB) is the repository for the three-dimensional structures of biological macromolecules, determined by experimental methods. The data in the archive are free and easily available via the Internet from any of the worldwide centers managing this global archive. These data are used by scientists, researchers, bioinformatics specialists, educators, students, and lay audiences to understand biological phenomena at a molecular level. Analysis of these structural data also inspires and facilitates new discoveries in science. This chapter describes the tools and methods currently used for deposition, processing, and release of data in the PDB. References to future enhancements are also included.

Eosinophil-derived neurotoxin (EDN) is a catalytically proficient member of the pancreatic ribonuclease superfamily secreted along with other eosinophil granule proteins during innate host defense responses and various eosinophil-related inflammatory and allergic diseases. The ribonucleolytic activity of EDN is central to its antiviral and neurotoxic activities and possibly to other facets of its biological activity. To probe the importance of this enzymatic activity further, specific inhibitors will be of great aid. Derivatives of 5'-ADP are among the most potent inhibitors currently known. Here, we use X-ray crystallography to investigate the binding of four natural nucleotides containing this moiety. 5'-ATP binds in two alternative orientations, one occupying the B2 subsite in a conventional manner and one being a retro orientation with no ordered adenosine moiety. Diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) bind with one adenine positioned at the B2 subsite, the polyphosphate chain extending across the P1 subsite in an ill-defined conformation, and a disordered second adenosine moiety. Diadenosine pentaphosphate (Ap5A), the most avid inhibitor of this series, binds in a completely ordered fashion with one adenine positioned conventionally at the B2 subsite, the polyphosphate chain occupying the P1 and putative P(-1) subsites, and the other adenine bound in a retro-like manner at the edge of the B1 subsite. The binding mode of each of these inhibitors has features seen in previously determined structures of adenosine diphosphates. We examine the structure-affinity relationships of these inhibitors and discuss the implications for the design of improved inhibitors.

The Macromolecular Structure Database (MSD) (http://www.ebi.ac.uk/msd/) [H. Boutselakis, D. Dimitropoulos, J. Fillon, A. Golovin, K. Henrick, A. Hussain, J. Ionides, M. John, P. A. Keller, E. Krissinel et al. (2003) E-MSD: the European Bioinformatics Institute Macromolecular Structure Database. Nucleic Acids Res., 31, 458-462.] group is one of the three partners in the worldwide Protein DataBank (wwPDB), the consortium entrusted with the collation, maintenance and distribution of the global repository of macromolecular structure data [H. Berman, K. Henrick and H. Nakamura (2003) Announcing the worldwide Protein Data Bank. Nature Struct. Biol., 10, 980.]. Since its inception, the MSD group has worked with partners around the world to improve the quality of PDB data, through a clean up programme that addresses inconsistencies and inaccuracies in the legacy archive. The improvements in data quality in the legacy archive have been achieved largely through the creation of a unified data archive, in the form of a relational database that stores all of the data in the wwPDB. The three partners are working towards improving the tools and methods for the deposition of new data by the community at large. The implementation of the MSD database, together with the parallel development of improved tools and methodologies for data harvesting, validation and archival, has lead to significant improvements in the quality of data that enters the archive. Through this and related projects in the NMR and EM realms the MSD continues to improve the quality of publicly available structural data.

The eosinophil major basic protein (EMBP), a constituent of the eosinophil secondary granule, is implicated in cytotoxicity and mediation of allergic disorders such as asthma. It is a member of the C-type lectin family, but lacks a Ca(2+)- and carbohydrate-binding site as seen in other members of this family. Here, we report the crystal structure of EMBP in complex with a heparin disaccharide and in the absence of Ca(2+), the first such report of any C-lectin with this sugar. We also provide direct evidence of binding of EMBP to heparin and heparin disaccharide by surface plasmon resonance. We propose that the sugars recognized by EMBP are likely to be proteoglycans such as heparin, leading to new interpretations for EMBP function.

The Macromolecular Structure Database (MSD) group (http://www.ebi.ac.uk/msd/) continues to enhance the quality and consistency of macromolecular structure data in the Protein Data Bank (PDB) and to work towards the integration of various bioinformatics data resources. We have implemented a simple form-based interface that allows users to query the MSD directly. The MSD 'atlas pages' show all of the information in the MSD for a particular PDB entry. The group has designed new search interfaces aimed at specific areas of interest, such as the environment of ligands and the secondary structures of proteins. We have also implemented a novel search interface that begins to integrate separate MSD search services in a single graphical tool. We have worked closely with collaborators to build a new visualization tool that can present both structure and sequence data in a unified interface, and this data viewer is now used throughout the MSD services for the visualization and presentation of search results. Examples showcasing the functionality and power of these tools are available from tutorial webpages (http://www. ebi.ac.uk/msd-srv/docs/roadshow_tutorial/).

The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the alpha-Gal epitope. alpha3GT catalyzes galactose transfer from UDP-Gal to beta-linked galactosides, such as lactose, and in the absence of an acceptor substrate, to water at a lower rate. We have used site-directed mutagenesis to investigate the roles in catalysis and specificity of residues in alpha3GT that form H-bonds as well as other interactions with substrates. Mutation of the conserved Glu(317) to Gln weakens lactose binding and reduces the k(cat) for galactosyltransfer to lactose and water by 2400 and 120, respectively. The structure is not perturbed by this substitution, but the orientation of the bound lactose molecule is changed. The magnitude of these changes does not support a previous proposal that Glu(317) is the catalytic nucleophile in a double displacement mechanism and suggests it acts in acceptor substrate binding and in stabilizing a cationic transition state for cleavage of the bond between UDP and C1 of the galactose. Cleavage of this bond also linked to a conformational change in the C-terminal region of alpha3GT that is coupled with UDP binding. Mutagenesis indicates that His(280), which is projected to interact with the 2-OH of the galactose moiety of UDP-Gal, is a key residue in the stringent donor substrate specificity through its role in stabilizing the bound UDP-Gal in a suitable conformation for catalysis. Mutation of Gln(247), which forms multiple interactions with acceptor substrates, to Glu reduces the catalytic rate of galactose transfer to lactose but not to water. This mutation is predicted to perturb the orientation or environment of the bound acceptor substrate. The results highlight the importance of H-bonds between enzyme and substrates in this glycosyltransferase, in arranging substrates in appropriate conformations and orientation for efficient catalysis. These factors are manifested in increases in catalytic rate rather than substrate affinity.

We have determined the structure of wild-type IP-10 from three crystal forms. The crystals provide eight separate models of the IP-10 chain, all differing substantially from a monomeric IP-10 variant examined previously by NMR spectroscopy. In each crystal form, IP-10 chains form conventional beta sheet dimers, which, in turn, form a distinct tetrameric assembly. The M form tetramer is reminiscent of platelet factor 4, whereas the T and H forms feature a novel twelve-stranded beta sheet. Analytical ultracentrifugation indicates that, in free solution, IP-10 exists in a monomer-dimer equilibrium with a dissociation constant of 9 microM. We propose that the tetrameric structures may represent species promoted by the binding of glycosaminoglycans. The binding sites for several IP-10-neutralizing mAbs have also been mapped.

Bovine alpha-1,3-galactosyltransferase (alpha3GT) catalyzes the synthesis of the alpha-galactose (alpha-Gal) epitope, the target of natural human antibodies. It represents a family of enzymes, including the histo blood group A and B transferases, that catalyze retaining glycosyltransfer reactions of unknown mechanism. An initial study of alpha3GT in a crystal form with limited resolution and considerable disorder suggested the possible formation of a beta-galactosyl-enzyme covalent intermediate (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638-649). Highly ordered structures are described for complexes of alpha3GT with donor substrate, UDP-galactose, UDP- glucose, and two acceptor substrates, lactose and N-acetyllactosamine, at resolutions up to 1.46 A. Structural and calorimetric binding studies suggest an obligatory ordered binding of donor and acceptor substrates, linked to a donor substrate-induced conformational change, and the direct participation of UDP in acceptor binding. The monosaccharide-UDP bond is cleaved in the structures containing UDP-galactose and UDP-glucose, producing non-covalent complexes containing buried beta-galactose and alpha-glucose. The location of these monosaccharides and molecular modeling suggest that binding of a distorted conformation of UDP-galactose may be important in the catalytic mechanism of alpha3GT.

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys(29) and with Cys(57) near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp(72), in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.

Human eosinophil-derived neurotoxin (EDN) is a small, basic protein that belongs to the ribonuclease A superfamily. EDN displays antiviral activity and causes the neurotoxic Gordon phenomenon when injected into rabbits. Although EDN and ribonuclease A have appreciable structural similarity and a conserved catalytic triad, their peripheral substrate-binding sites are not conserved. The crystal structure of recombinant EDN (rEDN) has been determined at 0.98 A resolution from data collected at a low temperature (100 K). We have refined the crystallographic model of the structure using anisotropic displacement parameters to a conventional R-factor of 0.116. This represents the highest resolution structure of rEDN determined to date and is only the second ribonuclease structure to be determined at a resolution greater than 1.0 A. The structure provides a detailed picture of the conformational freedom at the various subsites of rEDN, and the water structure accounts for more than 50% of the total solvent content of the unit cell. This information will be crucial for the design of tight-binding inhibitors to restrain the ribonucleolytic activity of rEDN.

UDP-galactose:beta-galactosyl alpha-1,3-galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-alpha-d-galactose into an alpha-1,3 linkage with beta-galactosyl groups in glycoconjugates. The enzyme is expressed in many mammalian species but is absent from humans, apes, and old world monkeys as a result of the mutational inactivation of the gene; in humans, a large fraction of natural antibodies are directed against its product, the alpha-galactose epitope. alpha3GT is a member of a family of metal-dependent retaining glycosyltransferases including the histo-blood group A and B synthases. A crystal structure of the catalytic domain of alpha3GT was recently reported (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638-649). However, because of the limited resolution (2.3 A) and high mobility of the atoms (as indicated by high B-factors) this structure (form I) does not provide a clear depiction of the catalytic site of the enzyme. Here we report a new, highly ordered structure for the catalytic domain of alpha3GT at 1.53-A resolution (form II). This provides a more accurate picture of the details of the catalytic site that includes a bound UDP molecule and a Mn(2+) cofactor. Significantly, in the new structure, the C-terminal segment (residues 358-368) adopts a very different, highly structured conformation and appears to form part of the active site. The properties of an Arg-365 to Lys mutant indicate that this region is important for catalysis, possibly reflecting its role in a donor substrate-induced conformational change.

The crystal structure of an antibacterial protein of immune origin (TSWAB), purified from tasar silkworm (Antheraea mylitta) larvae after induction by Escherichia coli infection, has been determined. This is the first insect lysozyme structure and represents induced lysozymes of innate immunity. The core structure of TSWAB is similar to c-type lysozymes and alpha-lactalbumins. However, TSWAB shows significant differences with respect to the other two proteins in the exposed loop regions. The catalytic residues in TSWAB are conserved with respect to the chicken lysozyme, indicating a common mechanism of action. However, differences in the noncatalytic residues in the substrate binding groove imply subtle differences in the specificity and the level of activity. Thus, conformational differences between TSWAB and chicken lysozyme exist, whereas functional mechanisms appear to be similar. On the other hand, alpha-lactalbumins and c-type lysozymes exhibit drastically different functions with conserved molecular conformation. It is evident that a common molecular scaffold is exploited in the three enzymes for apparently different physiological roles. It can be inferred on the basis of the structure-function comparison of these three proteins having common phylogenetic origin that the conformational changes in a protein are minimal during rapid evolution as compared with those in the normal course of evolution.

The eosinophil major basic protein (EMBP) is the predominant constituent of the crystalline core of the eosinophil primary granule. EMBP is directly implicated in epithelial cell damage, exfoliation, and bronchospasm in allergic diseases such as asthma. Here we report the crystal structure of EMBP at 1.8 A resolution, and show that it is similar to that of members of the C-type lectin superfamily with which it shares minimal amino acid sequence identity (approximately 15--28%). However, this protein lacks a Ca(2+)/carbohydrate-binding site. Our analysis suggests that EMBP specifically binds heparin. Based on our results, we propose a possible new function for this protein, which is likely to have implications for EMBP function.

Eosinophil-derived neurotoxin (EDN), a basic ribonuclease found in the large specific granules of eosinophils, belongs to the pancreatic RNase A family. Although its physiological function is still unclear, it has been shown that EDN is a neurotoxin capable of inducing the Gordon phenomenon in rabbits. EDN is also a potent helminthotoxin and can mediate antiviral activity of eosinophils against isolated virions of the respiratory syncytial virus. EDN is a catalytically efficient RNase sharing similar substrate specificity with pancreatic RNase A with its ribonucleolytic activity being absolutely essential for its neurotoxic, helminthotoxic, and antiviral activities. The crystal structure of recombinant human EDN in the unliganded form has been determined previously (Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. G. (1996) J. Mol. Biol. 260, 540-552). We have now determined high resolution (1.8 A) crystal structures for EDN in complex with adenosine-3',5'-diphosphate (3',5'-ADP), adenosine-2',5'-di-phosphate (2',5'-ADP), adenosine-5'-diphosphate (5'-ADP) as well as for a native structure in the presence of sulfate refined at 1.6 A. The inhibition constant of these mononucleotides for EDN has been determined. The structures present the first detailed picture of differences between EDN and RNase A in substrate recognition at the ribonucleolytic active site. They also provide a starting point for the design of tight-binding inhibitors, which may be used to restrain the RNase activity of EDN.

The angiogenic molecule placenta growth factor (PlGF) is a member of the cysteine-knot family of growth factors. In this study, a mature isoform of the human PlGF protein, PlGF-1, was crystallized as a homodimer in the crystallographic asymmetric unit, and its crystal structure was elucidated at 2.0 A resolution. The overall structure of PlGF-1 is similar to that of vascular endothelial growth factor (VEGF) with which it shares 42% amino acid sequence identity. Based on structural and biochemical data, we have mapped several important residues on the PlGF-1 molecule that are involved in recognition of the fms-like tyrosine kinase receptor (Flt-1, also known as VEGFR-1). We propose a model for the association of PlGF-1 and Flt-1 domain 2 with precise shape complementarity, consider the relevance of this assembly for PlGF-1 signal transduction, and provide a structural basis for altered specificity of this molecule.

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.