Synthesis and characterization of mono and polymeric cyclopalladated compounds: Crystal and molecular structure of [{Pd(dmba)(ONO2)}(2)(mu-pz)] center dot H2O (pz = pyrazine)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avrami exponent of crystallization in tellurite glasses

Nucleation process and crystal growth for three samples of the (20-x)Li(2)O-80TeO(2)-xWO(3) glass system were studied using X-ray diffraction and differential scanning calorimetry techniques. X-ray diffraction data confirmed the amorphous characteristic of the as-quenched samples and indicated the growth of crystalline phases formed due to the thermal treatment for annealed samples. These results reveal the presence of three distinct gamma-TeO(2), alpha-TeO(2) and alpha-Li(2)Te(2)O(5) crystalline phases in the TL sample, and two distinct alpha-TeO(2) and gamma-TeO(2) crystalline phases in the TLW5 and TLW10 samples. The activation energy and the Avrami exponent were determined from DSC measurements. The activation energy values X-ray diffraction data of the TLW10 glass sample suggest that gamma-TeO(2) phase occur before the alpha-TeO(2). The results obtained for the Avrami exponent point to that the nucleation process is volumetric and that the crystal growth is two or three-dimensional.

Crystal structure of myotoxin II, a monomeric Lys49-Phospholipase A(2) homologue isolated from the venom of Cerrophidion (Bothrops) godmani

Lys49-Phospholipase A(2) (Lys49-PLA(2)) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. With the aim of determining the structural basis for this novel activity, we have solved the crystal structure of myotoxin-II, a Lys49-PLA(2) isolated from the venom of Cerrophidion (Bothrops) godmani (godMT-II) at 2.8 Angstrom resolution by molecular replacement. The final model has been refined to a final crystallografic residual (R-factor) of 18.8% (R-free = 28.2%), with excellent stereochemistry. godMT-II is also monomeric in the crystalline state, and small-angle X-ray scattering results demonstrate that the protein is monomeric in solution under fisicochemical conditions similar to those used in the crystallographic studies. (C) 1999 Academic Press.

Structural insights for fatty acid binding in a Lys49-phospholipase A(2): crystal structure of myotoxin II from Bothrops molojeni complexed with stearic acid

The crystal structure of dimeric Lys49-phospholipase A2 myotoxin-II from Bothrops moojeni (MjTX-II) co-crystallized with stearic acid (C18H36O2) has been determined at a resolution of 1.8 angstrom. The electron density maps permitted the unambiguous inclusion of six stearic acid molecules in the refinement. Two stearic acid molecules could be located in the substrate-binding cleft of each monomer in positions, which favor the interaction of their carboxyl groups with active site residues. The way of binding of stearic acids to this Lys49-PLA(2)s is analogous to phospholipids and transition state analogues to catalytically active PLA(2)s. Two additional stearic acid molecules were located at the dimer interface region, defining a hitherto unidentified acyl-binding site on the protein surface. The strictly conserved Lys122 for Lys49-PLA(2)s may play a fundamental role for stabilization of legend-protein complex. The comparison of MjTX-II/satiric acid complex with other Lys-PLA(2)s structures whose putative fatty acids were located at their active site is also analysed. Molecular details of the stearic acid/protein interactions provide insights to binding in croup I/II PLA(2)s and to the possible interactions of Lys49-PLA(2)s with target membranes. (c) 2004 Elsevier SAS. All rights reserved.

Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A(2): quaternary structure and inhibition mechanism insights

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. A class of PLA(2)-like proteins has been described which despite PLA(2) activity on artificial substrate, due to a D49K mutation, is still highly myonecrotic. This work reports the X-ray structure determination of two Lys49-PLA(2)s from Bothrops neuwiedi pauloensis (BnSP-7 and BnSP-6) and, for the first time, the comparison of eight dimeric Lys49-PLA2s. This comparison reveals that there are not just two (open and closed) but at least six different conformations. The binding of fatty acid observed in three recent Lys49-PLA(2) structures seems to be independent of their quaternary conformation. Cys29 polarization by Lys122 is not significant for BnSP-7 and BnSP-6 or other structures not bound by fatty acids. These structures may be in an active state when nothing is bound to them and the Lys122/Cys29 interactions are weak or absent. (C) 2003 Elsevier B.V. All rights reserved.

Crystal structure of an acidic platelet aggregation inhibitor and hypotensive phospholipase A(2) in the monomeric and dimeric states: insights into its oligomeric state

Phospholipases A(2) belong to the superfamily of proteins which hydrolyzes the sn-2 acyl groups of membrane phospholipids to release arachidonic acid and lysophospholipids. An acidic phospholipase A(2) isolated from Bothrops juraracussu snake venom presents a high catalytic, platelet aggregation inhibition and hypotensive activities. This protein was crystallized in two oligomeric states: monomeric and dimeric. The crystal structures were solved at 1.79 and 1.90 Angstrom resolution, respectively, for the two states. It was identified a Na+ ion at the center of Ca2+-binding site of the monomeric form. A novel dimeric conformation with the active sites exposed to the solvent was observed. Conformational states of the molecule may be due to the physicochemical conditions used in the crystallization experiments. We suggest dimeric state is one found in vivo. (C) 2004 Elsevier B.V. All rights reserved.

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Cratalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

Crystal structure of a myotoxic Asp49-phospholipase A(2) with low catalytic activity: Insights into Ca2+ -independent catalytic mechanism

A myotoxic Asp49-phospholipase A(2) (Asp49-PLA(2)) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA(2)s. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA(2) from B. jararacussu) and other Asp49-PLA(2)s. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA(2)s, making a hydrogen bond with the atom 0 delta 2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na+ ions at the Ca2+-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA(2)s which are able to bind Ca2+ ions are also able to bind Na+ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA(2)s indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA(2)s. (c) 2008 Elsevier B.V All rights reserved.

Crystal structure of a phospholipase A(2) homolog complexed with p-bromophenacyl bromide reveals important structural changes associated with the inhibition of myotoxic activity

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

Crystal structure of human purine nucleoside phosphorylase complexed with acyclovir

In human, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This work reports the first crystallographic Study of human PNP complexed with acyclovir (HsPNP:Acy). Acyclovir is a potent clinically useful inhibitor of replicant herpes simplex virus that also inhibits human PNP but with a relatively lower inhibitory activity (K-i=90muM). Analysis of the structural differences among the HsPNP:Acy complex, PNP apoenzyme, and HsPNP:Immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design. (C) 2003 Published by Elsevier B.V.

Crystal structure of human PNP complexed with guanine

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. More recently, the 3-D structure of human PNP has been refined to 2.3 Angstrom resolution, which allowed a redefinition of the residues involved in the substrate-binding sites and provided a more reliable model for structure-based design of inhibitors. This work reports crystallographic study of the complex of Human PNP:guanine (HsPNP:Gua) solved at 2.7 Angstrom resolution using synchrotron radiation. Analysis of the structural differences among the HsPNP:Gua complex, PNP apoenzyme, and HsPNP:immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design. (C) 2003 Elsevier B.V. All rights reserved.

Crystal structure of human PNP complexed with hypoxanthine and sulfate ion

Purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme, which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. Human PNP has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Here we report the crystal structure of human PNP in complex with hypoxanthine, refined to 2.6 Angstrom resolution. The intermolecular interaction between ligand and PNP is discussed. (C) 2004 Elsevier B.V. All rights reserved.

Crystal structure of human purine nucleoside phosphorylase at 2.3 angstrom resolution

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. In human, PNP is the only route for degradation of deoxyguanosine and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and its low resolution structure has been used for drug design. Here we report the structure of human PNP solved to 2.3 Angstrom resolution using synchrotron radiation and cryocrystallographic techniques. This structure allowed a more precise analysis of the active site, generating a more reliable model for substrate binding. The higher resolution data allowed the identification of water molecules in the active site, which suggests binding partners for potential ligands. Furthermore, the present structure may be used in the new structure-based design of PNP inhibitors. (C) 2003 Published by Elsevier B.V.

Insights into metal ion binding in phospholipases A(2): ultra high-resolution crystal structures of an acidic phospholipase A(2) in the Ca2+ free and bound states

The electrophile Ca2+ is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca2+ free and bound states at 0.97 and 1.60 angstrom resolutions, respectively. In the Ca2+ bound state, the Ca2+ ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca2+, a water molecule occupies the position of the Ca2+ ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation. (c) 2005 Elsevier SAS. All rights reserved.