Catherine H. Schein

SDAP: database and computational tools for allergenic proteins

SDAP (Structural Database of Allergenic Proteins) is a web server that provides rapid, cross-referenced access to the sequences, structures and IgE epitopes of allergenic proteins. The SDAP core is a series of CGI scripts that process the user queries, interrogate the database, perform various computations related to protein allergenic determinants and prepare the output HTML pages. The database component of SDAP contains information about the allergen name, source, sequence, structure, IgE epitopes and literature references and easy links to the major protein (PDB, SWISS-PROT/TrEMBL, PIR-ALN, NCBI Taxonomy Browser) and literature (PubMed, MEDLINE) on-line servers. The computational component in SDAP uses an original algorithm based on conserved properties of amino acid side chains to identify regions of known allergens similar to user-supplied peptides or selected from the SDAP database of IgE epitopes. This and other bioinformatics tools can be used to rapidly determine potential cross-reactivities between allergens and to screen novel proteins for the presence of IgE epitopes they may share with known allergens. SDAP is available via the World Wide Web at http://fermi.utmb.edu/SDAP/.

InterProSurf : a web server for predicting interacting sites on protein surfaces

UNLABELLED A new web server, InterProSurf, predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. Here we illustrate the application of InterProSurf to determine which areas of Bacillus anthracis toxins and measles virus hemagglutinin protein interact with their respective cell surface receptors. The computationally predicted regions overlap with those regions previously identified as interface regions by sequence analysis and mutagenesis experiments. AVAILABILITY The InterProSurf web server is available at http://curie.utmb.edu/

Data mining of sequences and 3D structures of allergenic proteins.

MOTIVATION Many sequences, and in some cases structures, of proteins that induce an allergic response in atopic individuals have been determined in recent years. This data indicates that allergens, regardless of source, fall into discreet protein families. Similarities in the sequence may explain clinically observed cross-reactivities between different biological triggers. However, previously available allergy databases group allergens according to their biological sources, or observed clinical cross-reactivities, without providing data about the proteins. A computer-aided data mining system is needed to compare the sequential and structural details of known allergens. This information will aid in predicting allergenic cross-responses and eventually in determining possible common characteristics of IgE recognition. RESULTS The new web-based Structural Database of Allergenic Proteins (SDAP) permits the user to quickly compare the sequence and structure of allergenic proteins. Data from literature sources and previously existing lists of allergens are combined in a MySQL interactive database with a wide selection of bioinformatics applications. SDAP can be used to rapidly determine the relationship between allergens and to screen novel proteins for the presence of IgE or T-cell epitopes they may share with known allergens. Further, our novel similarity search method, based on five dimensional descriptors of amino acid properties, can be used to scan the SDAP entries with a peptide sequence. For example, when a known IgE binding epitope from shrimp tropomyosin was used as a query, the method rapidly identified a similar sequence in known shellfish and insect allergens. This prediction of cross-reactivity between allergens is consistent with clinical observations. AVAILABILITY SDAP is available on the web at http://fermi.utmb.edu/SDAP/index.html

Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3.

The Jun a 3 protein from mountain cedar (Juniperus ashei) pollen, a member of group 5 of the family of plant pathogenesis-related proteins (PR-proteins), reacts with serum IgE from patients with cedar hypersensitivity. We used the crystal structures of two other proteins of this group, thaumatin and an antifungal protein from tobacco, both approximately 50% identical in sequence to Jun a 3, as templates to build homology models for the allergen. The in-house programs EXDIS and FANTOM were used to extract distance and dihedral angle constraints from the Protein Data Bank files and determine energy-minimized structures. The mean backbone deviations for the energy-refined model structures from either of the templates is <1 A, their conformational energies are low, and their stereochemical properties (determined with PROCHECK) are acceptable. The circular dichroism spectrum of Jun a 3 is consistent with the postulated beta-sheet core. Tryptic fragments of Jun a 3 that reacted with IgE from allergic patients all mapped to one helical/loop surface of the models. The Jun a 3 models have features common to aerosol allergens from completely different protein families, suggesting that tertiary structural elements may mediate the triggering of an allergic response.

Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1.

BACKGROUND Cedar pollens cause allergic disease in diverse geographic areas. We have recently purified and characterized the major mountain cedar (Juniperus ashei) pollen allergen, Jun a 1. OBJECTIVE A full-length complementary DNA for Jun a 1 was cloned and sequenced, and the recombinant protein was expressed. METHODS Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primers based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by comparison with N-terminal amino acid sequencing of the intact tryptic fragments of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity was analyzed by Western blotting with patient sera. RESULTS Jun a 1 possesses a high level of amino acid sequence homology with Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase activity was identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 potential N-glycosylation sites that were distinct from those found in Cry j 1. The IgE from patient sera bound recombinant Jun a 1 in Western blot analysis. CONCLUSION The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glycosylation suggest little overlap of glycopeptide epitopes.

Characteristic motifs for families of allergenic proteins.

The identification of potential allergenic proteins is usually done by scanning a database of allergenic proteins and locating known allergens with a high sequence similarity. However, there is no universally accepted cut-off value for sequence similarity to indicate potential IgE cross-reactivity. Further, overall sequence similarity may be less important than discrete areas of similarity in proteins with homologous structure. To identify such areas, we first classified all allergens and their subdomains in the Structural Database of Allergenic Proteins (SDAP, http://fermi.utmb.edu/SDAP/) to their closest protein families as defined in Pfam, and identified conserved physicochemical property motifs characteristic of each group of sequences. Allergens populate only a small subset of all known Pfam families, as all allergenic proteins in SDAP could be grouped to only 130 (of 9318 total) Pfams, and 31 families contain more than four allergens. Conserved physicochemical property motifs for the aligned sequences of the most populated Pfam families were identified with the PCPMer program suite and catalogued in the webserver MotifMate (http://born.utmb.edu/motifmate/summary.php). We also determined specific motifs for allergenic members of a family that could distinguish them from non-allergenic ones. These allergen specific motifs should be most useful in database searches for potential allergens. We found that sequence motifs unique to the allergens in three families (seed storage proteins, Bet v 1, and tropomyosin) overlap with known IgE epitopes, thus providing evidence that our motif based approach can be used to assess the potential allergenicity of novel proteins.

A “moving metal mechanism” for substrate cleavage by the DNA repair endonuclease APE‐1

Apurinic/apyrimidinic endonuclease (APE‐1) is essential for base excision repair (BER) of damaged DNA. Here molecular dynamics (MD) simulations of APE1 complexed with cleaved and uncleaved damaged DNA were used to determine the role and position of the metal ion(s) in the active site before and after DNA cleavage. The simulations started from an energy minimized wild‐type structure of the metal‐free APE1/damaged‐DNA complex (1DE8). A grid search with one Mg2+ ion located two low energy clusters of Mg2+ consistent with the experimentally determined metal ion positions. At the start of the longer MD simulations, Mg2+ ions were placed at different positions as seen in the crystal structures and the movement of the ion was followed over the course of the trajectory. Our analysis suggests a “moving metal mechanism” in which one Mg2+ ion moves from the B‐ (more buried) to the A‐site during substrate cleavage. The anticipated inversion of the phosphate oxygens occurs during the in‐line cleavage reaction. Experimental results, which show competition between Ca2+ and Mg2+ for catalyzing the reaction, and high concentrations of Mg2+ are inihibitory, indicate that both sites cannot be simultaneously occupied for maximal activity. Proteins 2007.

Accounting for ligand-bound metal ions in docking small molecules on adenylyl cyclase toxins.

The adenylyl cyclase toxins produced by bacteria (such as the edema factor (EF) of Bacillus anthracis and CyaA of Bordetella pertussis) are important virulence factors in anthrax and whooping cough. Co‐crystal structures of these proteins differ in the number and positioning of metal ions in the active site. Metal ions bound only to the ligands in the crystal structures are not included during the docking. To determine what effect these “missing” metals have on docking results, the AutoDock, LigandFit/Cerius2, and FlexX programs were compared for their ability to correctly place substrate analogues and inhibitors into the active sites of the crystal structures of EF, CyaA, and mammalian adenylate cyclase. Protonating the phosphates of substrate analogues improved the accuracy of docking into the active site of CyaA, where the grid did not account for one of the three Mg2+ ions in the crystal structure. The AutoDock ranking (based on docking energies) of a test group of compounds was relatively unaffected by protonation of carboxyl groups. However, the ranking by FlexX‐ChemScore varied significantly, especially for docking to CyaA, suggesting that alternate protonation states should be tested when screening compound libraries with this program. When the charges on the bound metal were set correctly, AutoDock was the most reliable program of the three tested with respect to positioning substrate analogues and ranking compounds according to their experimentally determined ability to inhibit EF. Proteins 2007.

Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219–237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K m value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.

Major linear IgE epitopes of mountain cedar pollen allergen Jun a 1 map to the pectate lyase catalytic site.

Resolution of the 3D structures and IgE epitopes of allergens may identify common or conserved features of allergens. Jun a 1, the predominant allergen in mountain cedar pollen, was chosen as a model for identifying common structural and functional features among a group of plant allergens. In this study, synthetic, overlapping peptides of Jun a 1 and sera from patients allergic to mountain cedar pollen were used to identify linear epitopes. A 3D model of Jun a 1 was produced using the Bacillus subtiles pectate lyase (PL) as a template and validated with biophysical measurements. This allowed mappings of four IgE binding sites on Jun a 1. Two of the epitopes mapped to turns or loops on the surface of the model structure. The other two epitopes mapped to the beta-sheet region, homologous to the catalytic site of PL. This region of Jun a 1 is highly conserved in the group 1 allergens from other cedar trees as well as microbial PLs. The finding that two out of three major IgE epitopes map to highly conserved catalytic regions of group 1 cedar allergens may help to explain the high degree of cross-reactivity between cedar pollen allergens and might represent a pattern of reactivity common to other allergens with catalytic activity.