Assessment of 3D models for allergen research.

Allergenic proteins must crosslink specific IgE molecules, bound to the surface of mast cells and basophils, to stimulate an immune response. A structural understanding of the allergen-IgE interface is needed to predict cross-reactivities between allergens and to design hypoallergenic proteins. However, there are less than 90 experimentally determined structures available for the approximately 1500 sequences of allergens and isoallergens cataloged in the Structural Database of Allergenic Proteins. To provide reliable structural data for the remaining proteins, we previously produced more than 500 3D models using an automated procedure, with strict controls on template choice and model quality evaluation. Here, we assessed how well the fold and residue surface exposure of 10 of these models correlated with recently published experimental 3D structures determined by X-ray crystallography or NMR. We also discuss the impact of intrinsically disordered regions on the structural comparison and epitope prediction. Overall, for seven allergens with sequence identities to the original templates higher than 27%, the backbone root-mean square deviations were less than 2 Å between the models and the subsequently determined experimental structures for the ordered regions. Further, the surface exposure of the known IgE epitopes on the models of three major allergens, from peanut (Ara h 1), latex (Hev b 2), and soy (Gly m 4), was very similar to the experimentally determined structures. For the three remaining allergens with lower sequence identities to the modeling templates, the 3D folds were correctly identified. However, the accuracy of those models is not sufficient for a reliable epitope mapping.

Clostridial toxins: sensing a target in a hostile gut environment.

The current global outbreak of Clostridium difficile infection exemplifies the major public health threat posed by clostridial glucosylating toxins. In the western world, C. difficile infection is one of the most prolific causes of bacterial-induced diarrhea and potentially fatal colitis. Two pathogenic enterotoxins, TcdA and TcdB, cause the disease. Vancomycin and metronidazole remain readily available treatment options for C. difficile infection, but neither is fully effective as is evident by high clinical relapse and fatality rates. Thus, there is an urgent need to find an alternative therapy that preferentially targets the toxins and not the drug-resistant pathogen. Recently, we addressed these critical issues in a Nature Medicine letter, describing a novel host defense mechanism for subverting toxin virulence that we translated into prototypic allosteric therapy for C. difficile infection. In this addendum article, we provide a continued perspective of this antitoxin mechanism and consider the broader implications of therapeutic allostery in combating gut microbial pathogenesis.

New insights into the structure of (1→3,1→6)-ß-D-glucan side chains in the Candida glabrata cell wall.

ß-Glucan is a (1→3)-ß-linked glucose polymer with (1→6)-ß-linked side chains and a major component of fungal cell walls. ß-Glucans provide structural integrity to the fungal cell wall. The nature of the (1-6)-ß-linked side chain structure of fungal (1→3,1→6)-ß-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-ß-linked side chains of Candida glabrata using high-field NMR. The (1→6)-ß-linked side chains have an average length of 4 to 5 repeat units spaced every 21 repeat units along the (1→3)-linked polymer backbone. Computer modeling suggests that the side chains have a bent curve structure that allows for a flexible interconnection with parallel (1→3)-ß-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-ß-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity.

AllerML: markup language for allergens.

Many concerns have been raised about the potential allergenicity of novel, recombinant proteins into food crops. Guidelines, proposed by WHO/FAO and EFSA, include the use of bioinformatics screening to assess the risk of potential allergenicity or cross-reactivities of all proteins introduced, for example, to improve nutritional value or promote crop resistance. However, there are no universally accepted standards that can be used to encode data on the biology of allergens to facilitate using data from multiple databases in this screening. Therefore, we developed AllerML a markup language for allergens to assist in the automated exchange of information between databases and in the integration of the bioinformatics tools that are used to investigate allergenicity and cross-reactivity. As proof of concept, AllerML was implemented using the Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/) database. General implementation of AllerML will promote automatic flow of validated data that will aid in allergy research and regulatory analysis.

Differential high-affinity interaction of dectin-1 with natural or synthetic glucans is dependent upon primary structure and is influenced by polymer chain length and side-chain branching.

Glucans are structurally diverse fungal biopolymers that stimulate innate immunity and are fungal pathogen-associated molecular patterns. Dectin-1 is a C-type lectin-like pattern recognition receptor that binds glucans and induces innate immune responses to fungal pathogens. We examined the effect of glucan structure on recognition and binding by murine recombinant Dectin-1 with a library of natural product and synthetic (1-->3)-beta/(1-->6)-beta-glucans as well as nonglucan polymers. Dectin-1 is highly specific for glucans with a pure (1-->3)-beta-linked backbone structure. Although Dectin-1 is highly specific for (1-->3)-beta-d-glucans, it does not recognize all glucans equally. Dectin-1 differentially interacted with (1-->3)-beta-d-glucans over a very wide range of binding affinities (2.6 mM-2.2 pM). One of the most striking observations that emerged from this study was the remarkable high-affinity interaction of Dectin-1 with certain glucans (2.2 pM). These data also demonstrated that synthetic glucan ligands interact with Dectin-1 and that binding affinity increased in synthetic glucans containing a single glucose side-chain branch. We also observed differential recognition of glucans derived from saprophytes and pathogens. We found that glucan derived from a saprophytic yeast was recognized with higher affinity than glucan derived from the pathogen Candida albicans. Structural analysis demonstrated that glucan backbone chain length and (1-->6)-beta side-chain branching strongly influenced Dectin-1 binding affinity. These data demonstrate: 1) the specificity of Dectin-1 for glucans; 2) that Dectin-1 differentiates between glucan ligands based on structural determinants; and 3) that Dectin-1 can recognize and interact with both natural product and synthetic glucan ligands.

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/

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 inhibitory, indicate that both sites cannot be simultaneously occupied for maximal activity.

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.