Crystal structure of myotoxin-II: A myotoxic phospholipase A2 - Homologue from Bothrops moojeni venom.

The crystal structure of Myotoxin-II (MjTX-II), a Lys49 PLA(2)-homologue from Bothrops moojeni venom has been determined and refined at 2.0 Angstrom to a crystallographic residual of 19.7% (R-free = 28.1%). MjTX-II is a dimer in the crystal, with the monomers in the asymmetric unit related by a two-fold symmetry axis running through the dimer interface. The dimers of MjTX-II and the Lys49 PLA(2) from B. asper venom are similar, however the relative orientations of the monomers suggests a flexible dimer interface, which serves as a hinge between the two molecules.

cDNA cloning and 1.75 angstrom crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 +/- 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-lengthamino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 angstrom resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (beta alpha)(8) barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the alpha-crystallin family

The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein ( sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 angstrom resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 angstrom. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.

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.

Crystal structure of a novel myotoxic Arg49 phospholipase A(2) homolog (zhaoermiatoxin) from Zhaoermia mangshanensis snake venom: Insights into Arg49 coordination and the role of Lys122 in the polarization of the C-terminus

The venom of Zhaoermia mangshanensis, encountered solely in Mt Mang in China's Hunan Province, exhibits coagulant, phosphodiesterase, L-amino acid oxidase, kallikrein, phospholipase A(2) and myotoxic activities. The catalytically inactive PLA(2) homolog referred to as zhaoermiatoxin is highly myotoxic and displays high myonecrotic and edema activities. Zhaoermiatoxin possesses a molecular weight of 13,972 Da, consists of 121 amino-acid residues crosslinked by seven disulfide bridges and shares high sequence homology with Lys49-PLA(2)s from the distantly related Asian pitvipers. However, zhaoermiatoxin possesses an arginine residue at position 49 instead of a lysine, thereby suggesting a secondary Lys49 -> Arg substitution which results in a catalytically inactive protein. We have determined the first crystal structure of zhaoermiatoxin, an Arg49-PLA(2), from Zhaoermia mangshanensis venom at 2.05 A resolution, which represents a novel member of phospholipase A(2) family. In this structure, unlike the Lys49 PLA(2)s, the C-terminus is well ordered and an unexpected non-polarized state of the putative calcium-binding loop due to the flip of Lys122 towards the bulk solvent is observed. The orientation of the Arg-49 side chain results in a similar binding mode to that observed in the Lys49 PLA(2)s; however, the guadinidium group is tri-coordinated by carbonyl oxygen atoms of the putative calcium-binding loop, whereas the N zeta atom of lysine is tetra-coordinated as a result of the different conformation adopted by the putative calcium-binding loop. (c) 2008 Elsevier Ltd. All rights reserved.

Specific and nonspecific collapse in protein folding funnels

Experiments with fast folding proteins are beginning to address the relationship between collapse and folding. We investigate how different scenarios for folding can arise depending on whether the folding and collapse transitions are concurrent or whether a nonspecific collapse precedes folding. Many earlier studies have focused on the limit in which collapse is fast compared to the folding time; in this work we focus on the opposite limit where, at the folding temperature, collapse and folding occur simultaneously. Real proteins exist in both of these limits. The folding mechanism varies substantially in these two regimes. In the regime of concurrent folding and collapse, nonspecific collapse now occurs at a temperature below the folding temperature (but slightly above the glass transition temperature).

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. © 2005 Elsevier Inc. All rights reserved.

cDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed β(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-β-d-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα) 8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182. © 2006 The Authors.

Crystal structure of Jararacussin-I: The highly negatively charged catalytic interface contributes to macromolecular selectivity in snake venom thrombin-like enzymes

Snake venom serine proteinases (SVSPs) are hemostatically active toxins that perturb the maintenance and regulation of both the blood coagulation cascade and fibrinolytic feedback system at specific points, and hence, are widely used as tools in pharmacological and clinical diagnosis. The crystal structure of a thrombin-like enzyme (TLE) from Bothrops jararacussu venom (Jararacussin-I) was determined at 2.48 Å resolution. This is the first crystal structure of a TLE and allows structural comparisons with both the Agkistrodon contortrix contortrix Protein C Activator and the Trimeresurus stejnegeri plasminogen activator. Despite the highly conserved overall fold, significant differences in the amino acid compositions and three-dimensional conformations of the loops surrounding the active site significantly alter the molecular topography and charge distribution profile of the catalytic interface. In contrast to other SVSPs, the catalytic interface of Jararacussin-I is highly negatively charged, which contributes to its unique macromolecular selectivity. © 2012 The Protein Society.

Structural bases for a complete myotoxic mechanism: Crystal structures of two non-catalytic phospholipases A(2)-like from Bothrops brazili venom

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

Crystal structure of mature 2S albumin from Moringa oleifera seeds

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

Spectroscopic and Catalytic Characterization of a Functional (FeFeII)-Fe-III Biomimetic for the Active Site of Uteroferrin and Protein Cleavage

A mixed-valence complex, [Fe(III)Fe(II)L1(mu-OAc)(2)]BF4 center dot H2O, where the ligand H(2)L1 = 2-{[[3-[((bis-(pyridin-2-ylmethyl)amino)methyl)-2-hydroxy-5-methylbenzyl](pyridin-2-ylmethyl)amino]methyl]phenol}, has been studied with a range of techniques, and, where possible, its properties have been compared to those of the corresponding enzyme system purple acid phosphatase. The (FeFeII)-Fe-III and Fe-2(III) oxidized species were studied spectroelectrochemically. The temperature-dependent population of the S = 3/2 spin states of the heterovalent system, observed using magnetic circular dichroism, confirmed that the dinuclear center is weakly antiferromagnetically coupled (H = -2JS(1).S-2, where J = -5.6 cm(-1)) in a frozen solution. The ligand-to-metal charge-transfer transitions are correlated with density functional theory calculations. The (FeFeII)-Fe-III complex is electron paramagnetic resonance (EPR)-silent, except at very low temperatures (<2 K), because of the broadening caused by the exchange coupling and zero-field-splitting parameters being of comparable magnitude and rapid spin-lattice relaxation. However, a phosphate-bound Fe-2(III) complex showed an EPR spectrum due to population of the S-tot = 3 state (J= -3.5 cm(-1)). The phosphatase activity of the (FeFeII)-Fe-III complex in hydrolysis of bis(2,4-dinitrophenyl)phosphate (k(cat.) = 1.88 x 10(-3) s(-1); K-m = 4.63 x 10(-3) mol L-1) is similar to that of other bimetallic heterovalent complexes with the same ligand. Analysis of the kinetic data supports a mechanism where the initiating nucleophile in the phosphatase reaction is a hydroxide, terminally bound to Fe-III. It is interesting to note that aqueous solutions of [Fe(III)Fe(II)L1(mu-OAc)(2)](+) are also capable of protein cleavage, at mild temperature and pH conditions, thus further expanding the scope of this complex's catalytic promiscuity.

Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in the unprotonated form, the protein structure is very stable for ionic strengths ranging from 0 to 225 mM. Therefore, our findings support the hypothesis that the histidines constitute the hot points that induce configurational changes of E protein in acid pH, and give extra motivation to the development of new ideas for antivirus compound design.

Synthesis, Biological Evaluation, And Molecular Modeling of Chalcone Derivatives As Potent Inhibitors of Mycobacterium tuberculosis Protein Tyrosine Phosphatases (PtpA and PtpB)

Tuberculosis (TB) is a major infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization (WHO), about 1.8 million people die from TB and 10 million new cases are recorded each year. Recently, a new series of naphthylchalcones has been identified as inhibitors of Mtb protein tyrosine phosphatases (PTPs). In this work, 100 chalcones were designed, synthesized, and investigated for their inhibitory properties against MtbPtps. Structure-activity relationships (SAR) were developed, leading to the discovery of new potent inhibitors with IC50 values in the low-micromolar range. Kinetic studies revealed competitive inhibition and high selectivity toward the Mtb enzymes. Molecular modeling investigations were carried out with the aim of revealing the most relevant structural requirements underlying the binding affinity and selectivity of this series of inhibitors as potential anti-TB drugs.