Structural characterization of novel chemotactic and mastoparan peptides from the venom of the social wasp Agelaia pallipes pallipes by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise de inflamassoma induzido por urato monossódico em monócitos de gestantes portadoras de pré-eclampsia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purificação, imobilização e caracterização bioquímica de lipase produzida por Penicillium sect. Gracilenta CBMAI 1583 em cultivo submerso

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cupin: A candidate molecular structure for the Nep1-like protein family

Background: NEP1-like proteins (NLPs) are a novel family of microbial elicitors of plant necrosis. Some NLPs induce a hypersensitive-like response in dicot plants though the basis for this response remains unclear. In addition, the spatial structure and the role of these highly conserved proteins are not known.Results: We predict a 3d-structure for the beta-rich section of the NLPs based on alignments, prediction tools and molecular dynamics. We calculated a consensus sequence from 42 NLPs proteins, predicted its secondary structure and obtained a high quality alignment of this structure and conserved residues with the two Cupin superfamily motifs. The conserved sequence GHRHDWE and several common residues, especially some conserved histidines, in NLPs match closely the two cupin motifs. Besides other common residues shared by dicot Auxin-Binding Proteins (ABPs) and NLPs, an additional conserved histidine found in all dicot ABPs was also found in all NLPs at the same position.Conclusion: We propose that the necrosis inducing protein class belongs to the Cupin superfamily. Based on the 3d-structure, we are proposing some possible functions for the NLPs.

Intermolecular interactions and characterization of the novel factor Xa exosite involved in macromolecular recognition and inhibition: Crystal structure of human Gla-domainless factor Xa complexed with the anticoagulant protein NAPc2 from the hematophagous nematode Ancylostoma caninum

NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K-i = 8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty. (c) 2006 Elsevier Ltd. All rights reserved.

Active and exo-site inhibition of human factor Xa: Structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum

Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K-i = 43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 angstrom resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 angstrom) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex. (c) 2007 Elsevier Ltd. All rights reserved.

Structure of a calcium-independent phospholipase-like myotoxic protein from Bothrops asper venom

Myotoxin II, a myotoxic calcium-independent phospholipase-like protein isolated from the venom of Bothrops asper, possesses no detectable phospholipase activity. The crystal structure has been determined and refined at 2.8 Angstrom to an R factor of 16.5% (F>3 sigma) with excellent stereochemistry. Amino-acid differences between catalytically active phospholipases and myotoxin LI in the Ca2+-binding region, specifically the substitutions Tyr28-->Asn, Gly32-->Leu and Asp49-->Lys, result in an altered local conformation. The key difference is that the epsilon-amino group of Lys49 fills the site normally occupied by the calcium ion in catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus, myotoxin II is present as a dimer both in solution and in the crystalline state. The two molecules in the asymmetric unit are related by a nearly perfect twofold axis, yet the dimer is radically different from the dimer formed by the phospholipase from Crotalus atrox. Whereas in C. atrox the dimer interface occludes the active sites, in myotoxin II they are exposed to solvent.

Genetic structure of Blastocerus dichotomus populations in the Parana River basin (Brazil) based on protein variability

The population structure of 147 marsh deer (Blastocerus dichotomus) from three areas in the Parana River basin, Brazil, was studied by observing protein polymorphism at 17 loci. Six loci were polymorphic and 11 monomorphic. The proportion of polymorphic loci (P) was 35.29% and the average heterozygosity (H) was 6.31%. Wright's F-ST indicated that only 4.9% of the total variation in allelic frequencies was due to genetic differences between the three groups. The high value of F-IS (0.246) indicated inbreeding in the marsh deer. Genetic distance values (D = 0.014-0.051) showed little divergence between the three areas. We suggest that probable mechanisms accounting for the genetic structure are female phylopatry and polygyny and also that inbreeding has resulted from decreasing areas of wetland leading to isolation, overhunting, and diseases transmitted by cattle.

The plant energy-dissipating mitochondrial systems: Depicting the genomic structure and the expression profiles of the gene families of uncoupling protein and alternative oxidase in monocots and dicots

The simultaneous existence of alternative oxidases and uncoupling proteins in plants has raised the question as to why plants need two energy-dissipating systems with apparently similar physiological functions. A probably complete plant uncoupling protein gene family is described and the expression profiles of this family compared with the multigene family of alternative oxidases in Arabidopsis thaliana and sugarcane (Saccharum sp.) employed as dicot and monocot models, respectively. In total, six uncoupling protein genes, AtPUMP1-6, were recognized within the Arabidopsis genome and five (SsPUMP1-5) in a sugarcane EST database. The recombinant AtPUMP5 protein displayed similar biochemical properties as AtPUMP1. Sugarcane possessed four Arabidopsis AOx1-type orthologues (SsAOx1a-1d); no sugarcane orthologue corresponding to Arabidopsis AOx2-type genes was identified. Phylogenetic and expression analyses suggested that AtAOx1d does not belong to the AOx1-type family but forms a new (AOx3-type) family. Tissue-enriched expression profiling revealed that uncoupling protein genes were expressed more ubiquitously than the alternative oxidase genes. Distinct expression patterns among gene family members were observed between monocots and dicots and during chilling stress. These findings suggest that the members of each energy-dissipating system are subject to different cell or tissue/organ transcriptional regulation. As a result, plants may respond more flexibly to adverse biotic and abiotic conditions, in which oxidative stress is involved. © The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ab initio protein folding simulations using atomic burials as informational intermediates between sequence and structure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Crystal structure of Staphylococcus aureus exfoliative toxin D-like protein: structural basis for the high specificity of exfoliative toxins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)