The CATH classification revisited-architectures reviewed and new ways to characterize structural divergence in superfamilies

The latest version of CATH (class, architecture, topology, homology) (version 3.2), released in July 2008 (http://www.cathdb.info), contains 1 14215 domains, 2178 Homologous superfamilies and 1110 fold groups. We have assigned 20 330 new domains, 87 new homologous superfamilies and 26 new folds since CATH release version 3.1. A total of 28 064 new domains have been assigned since our NAR 2007 database publication (CATH version 3.0). The CATH website has been completely redesigned and includes more comprehensive documentation. We have revisited the CATH architecture level as part of the development of a `Protein Chart` and present information on the population of each architecture. The CATHEDRAL structure comparison algorithm has been improved and used to characterize structural diversity in CATH superfamilies and structural overlaps between superfamilies. Although the majority of superfamilies in CATH are not structurally diverse and do not overlap significantly with other superfamilies, similar to 4% of superfamilies are very diverse and these are the superfamilies that are most highly populated in both the PDB and in the genomes. Information on the degree of structural diversity in each superfamily and structural overlaps between superfamilies can now be downloaded from the CATH website.

Computational studies of the reversible domain swapping of p13suc1

A minimalist representation of protein structures using a Go- like potential for interactions is implemented to investigate the mechanisms of the domain swapping of p13suc1, a protein that exists in two native conformations: a monomer and a domain- swapped dimer formed by the exchange of a beta- strand. Inspired by experimental studies which showed a similarity of the transition states for folding of the monomer and the dimer, in this study we justify this similarity in molecular descriptions. When intermediates are populated in the simulations, formation of a domain- swapped dimer initiates from the ensemble of unfolded monomers, given by the fact that the dimer formation occurs at the folding/ unfolding temperature of the monomer ( T-f). It is also shown that transitions, leading to a dimer, involve the presence of two intermediates, one of them has a dimeric form and the other is monomeric; the latter is much more populated than the former. However, at temperatures lower than T-f, the population of intermediates decreases. It is argued that the two folded forms may coexist in absence of intermediates at a temperature much lower than T-f. Computational simulations enable us to find a mechanism, `` lock- and- dock'', for domain swapping of p13suc1. To explore the route toward dimer formation, the folding of unstructured monomers must be retarded by first locking one of the free ends of each chain. Then, the other free termini could follow and dock at particular regions, where most intrachain contacts are formed, and thus de. ne the transition states of the dimer. The simulations also showed that a decrease in the maximum distance between monomers increased their stability, which is explained based on confinement arguments. Although the simulations are based on models extracted from the native structure of the monomer and the dimer of p13suc1, the mechanism of the domain- swapping process could be general, not only for p13suc1.

Parmodel: a web server for automated comparative modeling of proteins

Parmodel is a web server for automated comparative modeling and evaluation of protein structures. The aim of this tool is to help inexperienced users to perform modeling, assessment, visualization, and optimization of protein models as well as crystallographers to evaluate structures solved experimentally. It is subdivided in four modules: Parmodel Modeling, Parmodel Assessment, Parmodel Visualization, and Parmodel Optimization. The main module is the Parmodel Modeling that allows the building of several models ford a same protein in a reduced time, through the distribution of modeling processes on a Beowulf cluster. Parmodel automates and integrates the main softwares used in comparative modeling as MODELLER, Whatcheck, Procheck, Raster3D, Molscript, and Gromacs. This web server is freely accessible at http://www.biocristalografia.df.ibilce.unesp.br/tools/parmodel. (C) 2004 Elsevier B.V. All rights reserved.

Estudos estruturais e cristalização de complexos formados entre toxinas botrópicas e inibidores vegetais

This research presents a study of structural complexes formed between the bothropstoxin BthTX-I and PrTX-I and phospholipase A2 (PLA2s) inhibitor 12- methoxy-4-methyl-voachalotine (MMV) of Tabernamontana catharinensis and rosmarinic acid (Cv-RA) of Rosmarinus officinalis. For both were used cocrystallization experiments with the toxins and inhibitors. In the future, the experimental three-dimensional structures of these complexes will be obtained through the technique of crystallography. Simultaneously, construction occurred in silico of inhibitors Cv-RA and MMV and attempts of docking between these binders and protein structures. These theoretical data provide important information regarding the nature of interactions between inhibitors, toxins and structural changes induced by the protein molecule inhibitors. Therefore, the acquisition of this information has great value to identification, characterization and development (drug design) of compounds with great potential for biotechnology and drug use. Considering further and more relevant aplications, these compounds could be used to control symptoms in many cases of ophidic poisoning and also in combating of pathological processes (degenerative inflammatory diseases and auto-immune diseases, Alzheimer's disease, schizophrenia, among others) whose causes are related to molecules belonging to the same groups of proteins which are classified bothropstoxin that were studied in this project

Looking over Toxin-K+ Channel Interactions. Clues from the Structural and Functional Characterization of alpha-KTx Toxin Tc32, a Kv1.3 Channel Blocker

alpha-KTx toxin Tc32, from the Amazonian scorpion Tityus cambridgei, lacks the dyad motif; including Lys27, characteristic of the family and generally associated with channel blockage. The toxin has been cloned and expressed for the first time. Electrophysiological experiments, by showing that the recombinant form blocks Kv1.3 channels of olfactory bulb periglomerular cells like the natural Tc32 toxin, when tested on the Kv1.3 channel of human T lymphocytes, confirmed it is in an active fold. The nuclear magnetic resonance-derived structure revealed it exhibits an alpha/beta scaffold typical of the members of the alpha-KTx family. TdK2 and TdK3, all belonging to the same alpha-KTx 18 subfamily, share significant sequence identity with Tc32 but diverse selectivity and affinity for Kv1.3 and Kv1.1 channels. To gain insight into the structural features that may justify those differences, we used the recombinant Tc32 nuclear magnetic resonance-derived structure to model the other two toxins, for which no experimental structure is available. Their interaction with Kv1.3 and Kv1.1 has been investigated by means of docking simulations. The results suggest that differences in the electrostatic features of the toxins and channels, in their contact surfaces, and in their total dipole moment orientations govern the affinity and selectivity of toxins. In addition, we found that, regardless of whether the dyad motif is present, it is always a Lys side chain that physically blocks the channels, irrespective of its position in the toxin sequence.

Structure determination of proteins and peptides in solution: simulation, chirality and NMR studies

The study of protein fold is a central problem in life science, leading in the last years to several attempts for improving our knowledge of the protein structures. In this thesis this challenging problem is tackled by means of molecular dynamics, chirality and NMR studies. In the last decades, many algorithms were designed for the protein secondary structure assignment, which reveals the local protein shape adopted by segments of amino acids. In this regard, the use of local chirality for the protein secondary structure assignment was demonstreted, trying to correlate as well the propensity of a given amino acid for a particular secondary structure. The protein fold can be studied also by Nuclear Magnetic Resonance (NMR) investigations, finding the average structure adopted from a protein. In this context, the effect of Residual Dipolar Couplings (RDCs) in the structure refinement was shown, revealing a strong improvement of structure resolution. A wide extent of this thesis is devoted to the study of avian prion protein. Prion protein is the main responsible of a vast class of neurodegenerative diseases, known as Bovine Spongiform Encephalopathy (BSE), present in mammals, but not in avian species and it is caused from the conversion of cellular prion protein to the pathogenic misfolded isoform, accumulating in the brain in form of amiloyd plaques. In particular, the N-terminal region, namely the initial part of the protein, is quite different between mammal and avian species but both of them contain multimeric sequences called Repeats, octameric in mammals and hexameric in avians. However, such repeat regions show differences in the contained amino acids, in particular only avian hexarepeats contain tyrosine residues. The chirality analysis of avian prion protein configurations obtained from molecular dynamics reveals a high stiffness of the avian protein, which tends to preserve its regular secondary structure. This is due to the presence of prolines, histidines and especially tyrosines, which form a hydrogen bond network in the hexarepeat region, only possible in the avian protein, and thus probably hampering the aggregation.

Composti organostannici nell'ambiente marino e membrane biologiche: risposte molecolari e biochimiche nei molluschi bivalvi

Organotin compounds are worldwide diffused environmental contaminants, mainly as consequence of their extensive past use as biocides in antifouling paints. In spite of law restrictions, due to unwanted effects, organotin still persist in waters, being poorly degraded, easily resuspended from sediments and bioaccumulated in exposed organisms. The widespread toxicity and the possible threat to humans, likely to be organotin-exposed through contaminated seafood, make organotin interactions with biomolecules an intriguing biochemical topic, apart from a matter of ecotoxicological concern. Among organotins, tributyltin (TBT) is long known as the most dangerous and abundant chemical species in the Mediterranean Sea. Due to its amphiphilic nature, provided by three lipophilic arms and an electrophilic tin core, TBT can be easily incorporated in biomembranes and affect their functionality. Accordingly, it is known as a membrane-active toxicant and a mitochondrial poison. Up to now the molecular action modes of TBT are still partially unclear and poorly explored in bivalve mollusks, even if the latter play a not neglectable role in the marine trophic chain and efficiently accumulate organotins. The bivalve mollusk Mytilus galloprovincialis, selected for all experiments, is widely cultivated in the Mediterranean and currently used in ecotoxicological studies. Most work of this thesis was devoted to TBT effects on mussel mitochondria, but other possible targets of TBT were also considered. A great deal of literature points out TBT as endocrine disrupter and the masculinization of female marine gastropods, the so-called imposex, currently signals environmental organotin contamination. The hormonal status of TBT-exposed mussels and the possible interaction between hormones and contaminants in modulating microsomal hydroxilases, involved in steroid hormone and organotin detoxification, were the research topics in the period spent in Barcelona (Marco Polo fellowship). The variegated experimental approach, which consisted of two exposure experiments and in vitro tests, and the choice of selected tissues of M. galloprovincialis, the midgut gland for mitochondrial and microsomal preparations for subsequent laboratory assays and the gonads for the endocrine evaluations, aimed at drawing a clarifying pattern on the molecular mechanisms involved in organotin toxicity. TBT was promptly incorporated in midgut gland mitochondria of adult mussels exposed to 0.5 and 1.0 μg/L TBT, and partially degraded to DBT. TBT incorporation was accompanied by a decrease in the mitochondrial oligomycin-sensitive Mg-ATPase activity, while the coexistent oligomycin-insensitive fraction was unaffected. Mitochondrial fatty acids showed a clear rise in n-3 polyunsaturated fatty acids after 120 hr of TBT exposure, mainly referable to an increase in 22:6 level. TBT was also shown to inhibit the ATP hydrolytic activity of the mitochondrial F1FO complex in vitro and to promote an apparent loss of oligomycin sensitivity at higher than 1.0 μM concentration. The complex dose-dependent profile of the inhibition curve lead to the hypothesis of multiple TBT binding sites. At lower than 1.0 μM TBT concentrations the non competitive enzyme inhibition by TBT was ascribed to the non covalent binding of TBT to FO subunit. On the other hand the observed drop in oligomycin sensitivity at higher than 1.0 μM TBT could be related to the onset of covalent bonds involving thiolic groups on the enzyme structure, apparently reached only at high TBT levels. The mitochondrial respiratory complexes were in vitro affected by TBT, apart from the cytocrome c oxidase which was apparently refractory to the contaminant. The most striking inhibitory effect was shown on complex I, and ascribed to possible covalent bonds of TBT with –SH groups on the enzyme complexes. This mechanism, shouldered by the progressive decrease of free cystein residues in the presence of increasing TBT concentrations, suggests that the onset of covalent tin-sulphur bonds in distinct protein structures may constitute the molecular basis of widespread TBT effects on mitochondrial complexes. Energy production disturbances, in turn affecting energy consuming mechanisms, could be involved in other cellular changes. Mussels exposed to a wide range of TBT concentrations (20 - 200 and 2000 ng/L respectively) did not show any change in testosterone and estrogen levels in mature gonads. Most hormones were in the non-biologically active esterified form both in control and in TBT-treated mussels. Probably the endocrine status of sexually mature mussels could be refractory even to high TBT doses. In mussel digestive gland the high biological variability of microsomal 7-benzyloxy-4-trifluoromethylcoumarin-O-Debenzyloxylase (BFCOD) activity, taken as a measure of CYP3A-like efficiency, probably concealed any enzyme response to TBT exposure. On the other hand the TBT-driven enhancement of BFCOD activity in vitro was once again ascribed to covalent binding to thiol groups which, in this case, would stimulate the enzyme activity. In mussels from Barcelona harbour, a highly contaminated site, the enzyme showed a decreased affinity for the 7-benzyloxy-4-trifluoromethylcoumarin (BCF) substrate with respect to mussel sampled from Ebro Delta, a non-polluted marine site. Contaminant exposure may thus alter the kinetic features of enzymes involved in detoxification mechanisms. Contaminants and steroid hormones were clearly shown to mutually interact in the modulation of detoxification mechanisms. The xenoestrogen 17α-ethylenyl estradiol (EE2) displayed a non-competitive mixed inhibition of CYP3A-like activity by a preferential bond to the free enzyme both in Barcelona harbour and Ebro Delta mussels. The possible interaction with co-present contaminants in Barcelona harbour mussels apparently lessened the formation of the ternary complex enzyme-EE2-BCF. The whole of data confirms TBT as membrane toxicant in mussels as in other species and stresses TBT covalent binding to protein thiols as a widespread mechanism of membrane-bound-enzyme activity modulation by the contaminant.

Structured antibody surfaces for bio-recognition and a label-free detection of bacteria

Antibody microarrays are of great research interest because of their potential application as biosensors for high-throughput protein and pathogen screening technologies. In this active area, there is still a need for novel structures and assemblies providing insight in binding interactions such as spherical and annulus-shaped protein structures, e.g. for the utilization of curved surfaces for the enhanced protein-protein interactions and detection of antigens. Therefore, the goal of the presented work was to establish a new technique for the label-free detection of bio-molecules and bacteria on topographically structured surfaces, suitable for antibody binding.rnIn the first part of the presented thesis, the fabrication of monolayers of inverse opals with 10 μm diameter and the immobilization of antibodies on their interior surface is described. For this purpose, several established methods for the linking of antibodies to glass, including Schiff bases, EDC/S-NHS chemistry and the biotin-streptavidin affinity system, were tested. The employed methods included immunofluorescence and image analysis by phase contrast microscopy. It could be shown that these methods were not successful in terms of antibody immobilization and adjacent bacteria binding. Hence, a method based on the application of an active-ester-silane was introduced. It showed promising results but also the need for further analysis. Especially the search for alternative antibodies addressing other antigens on the exterior of bacteria will be sought-after in the future.rnAs a consequence of the ability to control antibody-functionalized surfaces, a new technique employing colloidal templating to yield large scale (~cm2) 2D arrays of antibodies against E. coli K12, eGFP and human integrin αvβ3 on a versatile useful glass surface is presented. The antibodies were swept to reside around the templating microspheres during solution drying, and physisorbed on the glass. After removing the microspheres, the formation of annuli-shaped antibody structures was observed. The preserved antibody structure and functionality is shown by binding the specific antigens and secondary antibodies. The improved detection of specific bacteria from a crude solution compared to conventional “flat” antibody surfaces and the setting up of an integrin-binding platform for targeted recognition and surface interactions of eukaryotic cells is demonstrated. The structures were investigated by atomic force, confocal and fluorescence microscopy. Operational parameters like drying time, temperature, humidity and surfactants were optimized to obtain a stable antibody structure.

Drug hypersensitivity reactions involving skin

Immune reactions to drugs can cause a variety of diseases involving the skin, liver, kidney, lungs, and other organs. Beside immediate, IgE-mediated reactions of varying degrees (urticaria to anaphylactic shock), many drug hypersensitivity reactions appear delayed, namely hours to days after starting drug treatment, showing a variety of clinical manifestations from solely skin involvement to fulminant systemic diseases which may be fatal. Immunohistochemical and functional studies of drug-specific T cells in patients with delayed reactions confirmed a predominant role for T cells in the onset and maintenance of immune-mediated delayed drug hypersensitivity reactions (type IV reactions). In these reactions, drug-specific CD4+ and CD8+ T cells are stimulated by drugs through their T cell receptors (TCR). Drugs can stimulate T cells in two ways: they can act as haptens and bind covalently to larger protein structures (hapten-carrier model), inducing a specific immune response. In addition, they may accidentally bind in a labile, noncovalent way to a particular TCR of the whole TCR repertoire and possibly also major histocompatibility complex (MHC)-molecules - similar to their pharmacologic action. This seems to be sufficient to reactivate certain, probably in vivo preactivated T cells, if an additional interaction of the drug-stimulated TCR with MHC molecules occurs. The mechanism was named pharmacological interaction of a drug with (immune) receptor and thus termed the p-i concept. This new concept may explain the frequent skin symptoms in drug hypersensitivity to oral or parenteral drugs. Furthermore, the various clinical manifestations of T cell-mediated drug hypersensitivity may be explained by distinct T cell functions leading to different clinical phenotypes. These data allowed a subclassification of the delayed hypersensitivity reactions (type IV) into T cell reactions which, by releasing certain cytokines and chemokines, preferentially activate and recruit monocytes (type IVa), eosinophils (type IVb), or neutrophils (type IVd).

Molding a peptide into an RNA site by in vivo peptide evolution

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single α-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage λ N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial α-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an “RNA world.”

Ferredoxin and ferredoxin–heme maquettes

A 16-amino acid residue peptide derived from a consensus motif of natural ferredoxins incorporates a tetranuclear iron sulfur cluster under physiological conditions. Successful assembly of the [4Fe–4S]2+/1+ cluster within a monomeric peptide was demonstrated using size exclusion chromatography, UV-visible, visible CD, and cryogenic EPR spectroscopies. The robustness of [4Fe–4S]2+/1+ formation was tested using peptides with either the ligating cysteine exchanged for alanine or with the intervening amino acids replaced by glycine. The small size of the peptide allows for modular incorporation into more complex protein structures. In one larger structure, we describe a tetra-α-helix bundle that self-assembles both iron–sulfur clusters and hemes, thereby demonstrating feasibility for the general synthesis of maquettes containing multiple, juxtaposed redox cofactors. This is a motif common to the catalytic sites of native oxidoreductases.

Paired Ig-like receptor homologs in birds and mammals share a common ancestor with mammalian Fc receptors

Paired Ig-like receptors (PIR) that can reciprocally modulate cellular activation have been described in mammals. In the present study, we searched expressed sequence tag databases for PIR relatives to identify chicken expressed sequence tags predictive of ≈25% amino acid identity to mouse PIR. Rapid amplification of cDNA ends (RACE)-PCR extension of expressed sequence-tag sequences using chicken splenic cDNA as a template yielded two distinct cDNAs, the sequence analysis of which predicted protein products with related extracellular Ig-like domains. Chicken Ig-like receptor (CHIR)-A was characterized by its transmembrane segment with a positively charged histidine residue and short cytoplasmic tail, thereby identifying CHIR-A as a candidate-activating receptor. Conversely, CHIR-B was characterized by its nonpolar transmembrane segment and cytoplasmic tail with two immunoreceptor tyrosine-based inhibitory motifs, indicating that it may serve as an inhibitory receptor. The use of CHIR amino acid sequences in a search for other PIR relatives led to the recognition of mammalian Fc receptors as distantly related genes. Comparative analyses based on amino acid sequences and three-dimensional protein structures provided molecular evidence for common ancestry of the PIR and Fc receptor gene families.

VIDA: a virus database system for the organization of animal virus genome open reading frames

VIDA is a new virus database that organizes open reading frames (ORFs) from partial and complete genomic sequences from animal viruses. Currently VIDA includes all sequences from GenBank for Herpesviridae, Coronaviridae and Arteriviridae. The ORFs are organized into homologous protein families, which are identified on the basis of sequence similarity relationships. Conserved sequence regions of potential functional importance are identified and can be retrieved as sequence alignments. We use a controlled taxonomical and functional classification for all the proteins and protein families in the database. When available, protein structures that are related to the families have also been included. The database is available for online search and sequence information retrieval at http://www.biochem.ucl.ac.uk/bsm/virus_database/VIDA.html.

The invariant system of coordinates of antibody molecules: prediction of the "standard" C alpha framework of VL and VH domains.

A new approach of comparing protein structures that does not involve the procedure of superposition is suggested. An invariant system of coordinates for immunoglobulin molecules that is based on the geometrical symmetry inherent to the variable domain light-chain (VL)-heavy-chain (VH) complex is described. The coordinates of the Calpha atoms in 22 immunoglobulin structures are calculated in the invariant system of coordinates. We found that 76 identical positions in this Calpha framework are symmetrical about the twofold axis. Comparison of the identical positions in these molecules allows us to select 96 positions in the light chains and 87 positions in the heavy chains whose Calpha atom coordinates are approximately the same. To check whether the average coordinates of Calpha atoms in these positions complies with the stereochemical requirements, we calculated Calpha-Calpha distances. Seventy-three positions of the light chains and 72 positions of the heavy chains satisfy the Calpha-Calpha distance criterion. The Calpha atoms in these positions are used for constructing the "standard" Calpha framework of VL and VH complexes. The average coordinates of Calpha atoms are presented.