Tirso Pons

Homology modeling, model and software evaluation: three related resources.

MOTIVATION Homology modeling is rapidly becoming the method of choice for obtaining three-dimensional coordinates for proteins because genome projects produce sequences at a much higher rate than NMR and X-ray laboratories can solve the three-dimensional structures. The quality of protein models will not be immediately clear to novices and support with the evaluation seems to be needed. Expert users are sometimes interested in evaluating the quality of modeling programs rather than the quality of the models themselves. RESULTS Three servers have been made available to the scientific community: a homology modeling server, a model quality evaluation server and a server that evaluates models built of proteins for which the structure is already known, thereby implicitly evaluating the quality of the modeling program. AVAILABILITY The modeling-related servers and several structure analysis servers are freely available at http://swift.embl-heidelberg.de/servers/ CONTACT gert.vriend@embl-heidelberg.de

Crystal Structure of Levansucrase from the Gram- Negative Bacterium Gluconacetobacter Diazotrophicus.

The endophytic Gram-negative bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA, EC 2.4.1.10), which converts sucrose into fructooligosaccharides and levan. The enzyme is included in GH (glycoside hydrolase) family 68 of the sequence-based classification of glycosidases. The three-dimensional structure of LsdA has been determined by X-ray crystallography at a resolution of 2.5 A (1 A=0.1 nm). The structure was solved by molecular replacement using the homologous Bacillus subtilis (Bs) levansucrase (Protein Data Bank accession code 1OYG) as a search model. LsdA displays a five-bladed beta-propeller architecture, where the catalytic residues that are responsible for sucrose hydrolysis are perfectly superimposable with the equivalent residues of the Bs homologue. The comparison of both structures, the mutagenesis data and the analysis of GH68 family multiple sequences alignment show a strong conservation of the sucrose hydrolytic machinery among levansucrases and also a structural equivalence of the Bs levansucrase Ca2+-binding site to the LsdA Cys339-Cys395 disulphide bridge, suggesting similar fold-stabilizing roles. Despite the strong conservation of the sucrose-recognition site observed in LsdA, Bs levansucrase and GH32 family Thermotoga maritima invertase, structural differences appear around residues involved in the transfructosylation reaction.

Three acidic residues are at the active site of a β‐propeller architecture in glycoside hydrolase families 32, 43, 62, and 68

Multiple‐sequence alignment of glycoside hydrolase (GH) families 32, 43, 62, and 68 revealed three conserved blocks, each containing an acidic residue at an equivalent position in all the enzymes. A detailed analysis of the site‐directed mutations so far performed on invertases (GH32), arabinanases (GH43), and bacterial fructosyltransferases (GH68) indicated a direct implication of the conserved residues Asp/Glu (block I), Asp (block II), and Glu (block III) in substrate binding and hydrolysis. These residues are close in space in the 5‐bladed β‐propeller fold determined for Cellvibrio japonicus α‐L‐arabinanase Arb43A [Nurizzo et al., Nat Struct Biol 2002;9:665–668] and Bacillus subtilis endo‐1,5‐α‐L‐arabinanase. A sequence–structure compatibility search using 3D‐PSSM, mGenTHREADER, INBGU, and SAM‐T02 programs predicted indistinctly the 5‐bladed β‐propeller fold of Arb43A and the 6‐bladed β‐propeller fold of sialidase/neuraminidase (GH33, GH34, and GH83) as the most reliable topologies for GH families 32, 62, and 68. We conclude that the identified acidic residues are located at the active site of a β‐propeller architecture in GH32, GH43, GH62, and GH68, operating with a canonical reaction mechanism of either inversion (GH43 and likely GH62) or retention (GH32 and GH68) of the anomeric configuration. Also, we propose that the β‐propeller architecture accommodates distinct binding sites for the acceptor saccharide in glycosyl transfer reaction. Proteins 2004.

Physico-chemical studies of molecular interactions between non-ionic surfactants and bovine serum albumin.

Surfactants, particularly non-ionic types, are often added to prevent and/or minimize protein aggregation during fermentation, purification, freeze-drying, shipping, and/or storage. In this work we have investigated the interactions between two non-ionic surfactants (Tween 20 and Tween 80) and bovine serum albumin (BSA), as model protein, using surface tension, fluorescence measurements and computational analysis. The results showed that, in both cases, the surface tension profile of the surfactants curve is modified upon addition of the protein, and the CMC values of Tween 20 and Tween 80 in the presence of protein are higher than the CMC values of the pure surfactants. The results indicate that although Tween 20 and Tween 80 do not greatly differ in their chemical structures, their interactions with BSA are of different nature, with distinct binding sites. Measurements at different protein concentrations showed that the interactions are also dependent on the protein aggregation state in solution. It was found from fluorescence studies that changes observed in both the intensity and wavelength of the tryptophan emission are probably caused by modifications of tryptophan environment due to surfactant binding, rather than by direct interaction. Based on a computational analysis of a BSA three-dimensional model, we hypothesize about the binding mechanism of non-ionic surfactant to globular protein, which allowed us to explain surface tension profiles and fluorescence results.

Towards a detailed atlas of protein–protein interactions

Protein interaction maps are the key to understand the complex world of biological processes inside the cell. Public protein databases have already catalogued hundreds of thousands of experimentally discovered interactions, and struggle to curate all the existing information dispersed through the literature. However, to be most efficient, standard protocols need to be implemented for direct submission of new interaction sets directly into databases. At the same time, great efforts are invested to expand the coverage of the interaction space and unveil the molecular details of such interactions up to the atomistic level. The net result will be the definition of a detailed atlas spanning the universe of protein interactions to guide the everyday work of the biologist.

Structural model for family 32 of glycosyl‐hydrolase enzymes

A structural model is presented for family 32 of the glycosyl‐hydrolase enzymes based on the beta‐propeller fold. The model is derived from the common prediction of two different threading methods, TOPITS and THREADER. In addition, we used a correlated mutation analysis and prediction of active‐site residues to corroborate the proposed model. Physical techniques (circular dichroism and differential scanning calorimetry) confirmed two aspects of the prediction, the proposed all‐beta fold and the multi‐domain structure. The most reliable three‐dimensional model was obtained using the structure of neuraminidase (1nscA) as template. The analysis of the position of the active site residues in this model is compatible with the catalytic mechanism proposed by Reddy and Maley (J. Biol. Chem. 271:13953–13958, 1996), which includes three conserved residues, Asp, Glu, and Cys. Based on this analysis, we propose the participation of one more conserved residue (Asp 162) in the catalytic mechanism. The model will facilitate further studies of the physical and biochemical characteristics of family 32 of the glycosyl‐hydrolases. Proteins 33:383–395, 1998.

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

Substitution of Asp-309 by Asn in the Arg-Asp-Pro (RDP) motif of Acetobacter diazotrophicus levansucrase affects sucrose hydrolysis, but not enzyme specificity.

beta-Fructofuranosidases share a conserved aspartic acid-containing motif (Arg-Asp-Pro; RDP) which is absent from alpha-glucopyranosidases. The role of Asp-309 located in the RDP motif of levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 was studied by site-directed mutagenesis. Substitution of Asp-309 by Asn did not affect enzyme secretion. The kcat of the mutant levansucrase was reduced 75-fold, but its Km was similar to that of the wild-type enzyme, indicating that Asp-309 plays a major role in catalysis. The two levansucrases showed optimal activity at pH 5.0 and yielded similar product profiles. Thus the mutation D309N affected the efficiency of sucrose hydrolysis, but not the enzyme specificity. Since the RDP motif is present in a conserved position in fructosyltransferases, invertases, levanases, inulinases and sucrose-6-phosphate hydrolases, it is likely to have a common functional role in beta-fructofuranosidases.

Beta-propellers: associated functions and their role in human diseases.

The beta-propeller fold appears as a very fascinating architecture based on four-stranded antiparallel and twisted beta-sheets, radially arranged around a central tunnel. Similar to the alpha/beta-barrel (TIM-barrel) fold, the beta-propeller has a wide range of different functions, and is gaining substantial attention. Some proteins containing beta-propeller domains have been implicated in the pathogenesis of a variety of diseases such as cancer, Alzheimer, Huntington, arthritis, familial hypercholesterolemia, retinitis pigmentosa, osteogenesis, hypertension, and microbial and viral infections. This article reviews some aspects of 3D structure, amino acids sequence regularities, and biological functions of the proteins containing beta-propeller domains. Major emphasis has been laid on beta-propellers whose functions are associated to human diseases. Recent research efforts reported in the fields of protein engineering, drug design, and protein structure-function relationship studies, concerning the beta-propeller architecture, have also been discussed.

A novel sea anemone peptide that inhibits acid-sensing ion channels

Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.