Clinical trials of newly developed anti-cancer agents in children with acute leukemia.
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Acute Disease↗
Biomedical subjects
T P Singh
Publications and source records attributed to T P Singh.
Specific binding of non-steroidal anti-inflammatory drugs (NSAIDs) to phospholipase A2: structure of the complex formed between phospholipase A2 and diclofenac at 2.7 A resolution.
Type IIA secretory phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids to release fatty acids and lysophospholipids. In order to elucidate the role of PLA2 in inflammatory disorders and to determine the mode of binding of non-steroidal anti-inflammatory drugs (NSAIDs) to PLA2, the detailed three-dimensional structure of a complex formed between a group IIA PLA2 from Daboia russelli pulchella and 2-[(2,6-dichlorophenyl)amino]benzeneacetic acid (diclofenac) has been determined. The preformed complex was crystallized by equilibrating the protein solution against a mixture of 0.20 M ammonium sulfate and 30% PEG 4000. The crystals belong to space group P4(3), with unit-cell parameters a = b = 53.0, c = 48.4 A. The structure was solved by the molecular-replacement method and refined to R(cryst) and R(free) factors of 0.192 and 0.211, respectively, using reflections to 2.7 A resolution. The structure showed that diclofenac occupies a very favourable position in the centre of the substrate-binding hydrophobic channel that allows a number of intermolecular interactions. The binding mode of diclofenac involved crucial interactions with important residues for substrate recognition such as Asp49, His48 and Gly30. In addition, it included three new interactions involving its Cl atoms with Phe5, Ala18 and Tyr22. It also showed an extensive network of hydrophobic interactions involving almost all of the residues of the substrate-binding hydrophobic channel. The binding affinity of diclofenac was determined using surface plasmon resonance, which gave an equilibrium constant of 4.8 +/- 0.2 x 10(-8) M.
Animals↗
Crystal structure of a novel phospholipase A2 from Naja naja sagittifera with a strong anticoagulant activity.
This is the first PLA(2) crystal structure from group I that shows a strong anticoagulant property. The monomeric PLA(2) was purified from the venom of Naja naja sagittifera (Indian cobra). Its amino acid sequence has been determined using cDNA technique. The amino acid sequence of sPLA(2) contains three positively charged and two negatively charged residues in the segment 54-71 (numbering scheme of sPLA(2)) thus giving this region an overall cationic amphiphilic surface. This suggested the presence of an anticoagulant activity in sPLA(2). The enzyme was crystallized using hanging drop vapour diffusion method in the presence of calcium chloride. The crystals belong to space group P4(1) with cell dimensions of a=b=42.0A, c=65.9A. The X-ray crystal structure was determined at 1.8A resolution using molecular replacement method and refined to an R value of 0.179 for 10,023 reflections. The overall scaffolding of sPLA(2) is essentially similar to those observed for other group I PLA(2)s. However, the conformations of various surface loops were found to be significantly different. The most significant observation pertains to the anticoagulant loop in which both the acidic residues are engaged in intramolecular interactions whereas all the three basic residues are free to interact with other molecules. This makes the sPLA(2) a potentially strong anticoagulating molecule.
Amino Acid Sequence↗
High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin.
Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 A, alpha = beta = 90, gamma = 120 degrees. This indicated a Matthews coefficient (VM) of 3.3 A3 Da(-1), corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 A resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.
Animals↗
Crystal structure of a calcium-induced dimer of two isoforms of cobra phospholipase A2 at 1.6 A resolution.
The calcium-induced formation of a complex between two isoforms of cobra venom phospholipase A2 reveals a novel interplay between the monomer-dimer and activity-inactivity transitions. The monodispersed isoforms lack activity in the absence of calcium ions while both molecules gain activity in the presence of calcium ions. At concentrations higher than 10 mg/ml, in the presence of calcium ions, they dimerize and lose activity again. The present study reports the crystal structure of a calcium-induced dimer between two isoforms of cobra phospholipase A2. In the complex, one molecule contains a calcium ion in the calcium binding loop while the second molecule does not possess an intramolecular calcium ion. However, there are two calcium ions per dimer in the structure. The second calcium ion is present at an intermolecular site and that is presumably responsible for the dimerization. The calcium binding loops of the two molecules adopt strikingly different conformations. The so-called calcium binding loop in the calcium-containing molecule adopts a normal conformation as generally observed in other calcium containing phospholipase A(2) enzymes while the conformation of the corresponding loop in the calcium free monomer deviates considerably with the formation of a unique intraloop Gly33 (N)-Cys27 (O) = 2.74 A backbone hydrogen bond. The interactions of Arg31 (B) with Asp49 (A) and absence of calcium ion are responsible for the loss of catalytic activity in molecule A while interactions of Arg2 (B) with Tyr52 (B) inactivate molecule B.
Animals↗
Crystal structure of a carbohydrate induced homodimer of phospholipase A2 from Bungarus caeruleus at 2.1A resolution.
This is the first crystal structure of a carbohydrate induced dimer of phospholipase A(2) (PLA(2)). This is an endogenous complex formed between two PLA(2) molecules and two mannoses. It was isolated from Krait venom (Bungarus caeruleus) and crystallized as such. The complete amino acid sequence of PLA(2) was determined using cDNA method. Three-dimensional structure of the complex has been solved with molecular replacement method and refined to a final R-factor of 0.192 for all the data in the resolution range 20.0-2.1A. The presence of mannose molecules in the protein crystals was confirmed using dinitrosalicylic acid test and the molecular weight of the dimer was verified with MALDI-TOF. As indicated by dynamic light scattering and analytical ultracentrifugation the dimer was also stable in solution. The good quality non-protein electron density at the interface of two PLA(2) molecules enabled us to model two mannoses. The mannoses are involved extensively in interactions with protein atoms of both PLA(2) molecules. Some of the critical amino acid residues such as Asp 49 and Tyr 31, which are part of the substrate-binding site, are found facing the interface and interacting with mannoses. The structure of the complex clearly shows that the dimerization is caused by mannoses and it results in the loss of enzymatic activity.
Amino Acid Sequence↗
Crystal structure of the complex of group I PLA2 with a group II-specific peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) at 2.6 A resolution.
Phospholipases A(2)s (PLA(2)s) are widely distributed in mammals and snake venoms. They catalyze the production of arachidonic acid from membrane phospholipids leading to the bioynthesis of pro-inflammatory eicosanoids. A peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) was designed and synthesized as a specific inhibitor of PLA(2). It was shown earlier that the peptide bound to group II PLA(2) specifically and had a dissociation constant (K(d)) of 8.8 x 10(-9) M. In the present studies for the binding of LAIYS with a group I PLA(2) from Naja naja sagittifera using surface plasmon resonance the dissociation constant was found to be 4.5 x 10(-5) M which is considerably lower than the value found for the group II PLA(2). In order to determine the details of binding at the molecular level, a group I PLA(2) from the venom of Naja naja sagittifera was crystallized with peptide LAIYS. The crystal structure showed the presence of LAIYS at the substrate-binding site but has fewer interactions than those observed with group II PLA(2) from Daboia russelli pulchella. The observed difference in the binding affinity is caused primarily due to poor fitting of the peptide LAIYS in the binding site of group I PLA(2). Apparently, the location of Trp 19 in group I PLA(2) is not favourable for the binding of LAIYS. The two complexes also differ drastically in the formation of intermolecular interactions. In the present structure, the side chain of Ser (P) interacts with His 48 and Asp 49 while in the complex with group II PLA(2) it was Tyr (P) OH that formed the corresponding interactions. Tyr (P) in group I PLA(2) is the main contributor of the hydrophobic interactions whereas in the complex of LAIYS with group II PLA(2) it was the peptide segment Leu-Ala-Ile that produced the bulk of hydrophobic forces. The structures further showed that the peptide LAIYS was fully inside the substrate-binding region of the group II PLA(2) while a significant portion of the peptide LAIYS was hanging outside the surface of the group I PLA(2). The buried area in the complex with group II PLA(2) was 811 A(2) whereas, the corresponding area in group I PLA(2) was 449 A(2). This shows that the peptide LAIYS is very compatible with the substrate-binding site of group II PLA(2) and rather poorly fits into the substrate-binding site of group I PLA(2). This indicates that a highly specific ligand for one form of PLA(2) may be a poor partner for another form of enzyme.
Binding Sites↗
Effects of branched beta-carbon dehydro-residues on peptide conformations: syntheses, crystal structures and molecular conformations of two tetrapeptides: (a) N-(benzyloxycarbonyl)-DeltaVal-Leu-DeltaPhe-Leu-OCH3 and (b) N-(benzyloxycarbonyl)-DeltaIle-Ala-DeltaPhe-Ala-OCH3.
The roles of branched beta-carbon dehydro-residues in the design of peptide conformations have not been systematically explored so far. In order to determine the effects of branched beta-carbon dehydro-residues on the peptide conformations, two N-protected tetrapeptides containing new combinations of DeltaVal and DeltaPhe in (a) N-(benzyloxycarbonyl)-DeltaVal-Leu-DeltaPhe-Leu-OCH(3) and DeltaIle and DeltaPhe in (b) N-(benzyloxycarbonyl)-DeltaIle-Ala-DeltaPhe-Ala-OCH(3) were synthesized by solution procedure. The crystal structures of these peptides were determined by X-ray diffraction methods. Single crystals of both peptides were grown by slow evaporation method from their solutions in acetone-water mixtures (80 : 20) at 25 degrees C. The crystals of these peptides belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 12.342(1) A, b = 15.659(1) A, c = 18.970(1) A for peptide (a) and a = 8.093(1) A, b = 15.791(1) A, c = 23.816(1) A for peptide (b) having Z = 4 in the unit cells of both peptides. The structures were refined by full-matrix least-squares procedure to R-factors of 0.076 and 0.052 respectively. Both peptides adopt the right-handed 3(10)-helical conformations stabilized by two intramolecular (i + 3-->i) hydrogen bonds between the CO of N-terminal benzyloxycarbonyl (Cbz) group and the NH of residue at position 3, and between the CO of residue at position 1 and NH of the residue at position 4. The two consecutive 10-membered rings formed by the hydrogen bonds have dihedral angles corresponding to the standard values for type III beta-turns. DeltaVal and DeltaIle in peptides (a) and (b) respectively are located at the (i + 1) position of the first beta-turn while DeltaPhe is located at the (i + 2) position of the second beta-turn. In the crystals, the molecules are linked head to tail by intermolecular hydrogen bonds to form long helical chains. The axes of helices are parallel to the b-axes while the neighbouring helices run in the opposite directions. The crystal packings are further stabilized by van der Waals forces between the columns of molecular packings.
Amino Acid Sequence↗
Sequence-induced trimerization of phospholipase A2: structure of a trimeric isoform of PLA2 from common krait (Bungarus caeruleus) at 2.5 A resolution.
The venom of the common Indian krait (Bungarus caeruleus) contains about a dozen isoforms of phospholipase A2 (PLA2), which exist in different oligomeric forms as well as in complexes with low-molecular-weight ligands. The basic objective of multimerization and complexation is either to inactivate PLA2 in the venom for long-term storage, to generate a new PLA2 function or to make a more lethal assembly. The current isoform was isolated from the venom of B. caeruleus. Dynamic light-scattering studies indicated the presence of a stable trimeric association of this PLA2. Its primary sequence was determined by cDNA cloning. The purified protein was crystallized with 2.8 M NaCl as a precipitating agent using the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 80.9, b = 80.5, c = 57.1 A, beta = 90.3 degrees. The structure was refined to a final R factor of 0.198. This is a novel trimeric PLA2 structure in which the central pore formed by the association of three molecules is filled with water molecules. The interactions across the pore take place via multiple water bridges primarily to the side chains of Arg, Lys and Thr residues. Approximately 12% of the total solvent-accessible surface area is buried in the core of the trimer. The active sites of all three molecules are located on the surface and are fully exposed to the solvent, resulting in a highly potent enzymatic unit.
Amino Acid Sequence↗
Crystal structure of echicetin from Echis carinatus (Indian saw-scaled viper) at 2.4A resolution.
Echicetin is a heterodimeric protein from the venom of the Indian saw-scaled viper, Echis carinatus. It binds to platelet glycoprotein Ib (GPIb) and thus inhibits platelet aggregation. It has two subunits, alpha and beta, consisting of 131 and 123 amino acid residues, respectively. The two chains are linked with a disulphide bond. The level of amino acid sequence homology between two subunits is 50%. The protein was purified from the venom of E.carinatus and crystallized using ammonium sulphate as a precipitant. The crystal structure has been determined at 2.4A resolution and refined to an R-factor of 0.187. Overall dimensions of the heterodimer are approximately 80Ax35Ax35A. The backbone folds of the two subunits are similar. The central portions of the polypeptide chains of alpha and beta-subunits move into each other to form a tight dimeric association. The remaining portions of the chains of both subunits fold in a manner similar to those observed in the carbohydrate-binding domains of C-type lectins. In echicetin, the Ca(2+)-binding sites are not present, despite being topologically equivalent to other similar Ca(2+)-binding proteins of the superfamily. The residues Ser41, Glu43 and Glu47 in the calcium-binding proteins of the related family are conserved but the residues Glu126/120 are replaced by lysine at the corresponding sites in the alpha and beta-subunits.
Base Sequence↗
Biotinyl endothelin-1 binding to endothelin receptor and its applications.
The endothelin (ET) system consists of two membrane receptor types A and B and three 21-mer isopeptides endothelin-1, endothelin-2, and endothelin-3 as ligands. This system is involved in many physiological processes such as vasomodulation, neurotransmission, embryonic development, renal function, and regulation of cell proliferation. In many pathophysiological conditions involving endothelin system, the endothelin antagonism could be a possible clinical treatment. Designing of an antagonist involves the characterization of the binding of the test compounds to the endothelin receptors. This is being carried out using radioactive ligand. A simpler and quicker method will be of great advantage. This study reports a non-radioactive method for establishing the IC50 concentrations of the ligand. This method uses biotinylated-endothelin-1 and streptavidin conjugated with horseradish peroxidase. Hydroxyl apatite gel is used for separating the bound and unbound biotin-tagged endothelin-1. This method is applicable to detergent solubilized receptors and purified recombinant receptors. The endothelin receptor type A expressed in Pichia pastoris system has been used in this study. We show that this method is applicable in Western blot analysis of endothelin-1 and its receptor complex. This can be used to localize the receptor molecules as well.
Animals↗
Structure of an acidic phospholipase A2 from Indian saw-scaled viper (Echis carinatus) at 2.6 A resolution reveals a novel intermolecular interaction.
The crystal structure of an acidic phospholipase A(2) from the venom of Echis carinatus (saw-scaled viper; scPLA(2)) has been determined at 2.6 A resolution and refined to a crystallographic R factor of 0.192. Although the overall structure of scPLA(2) is essentially similar to those of other group II acidic PLA(2)s from different species, it shows unique features in several parts. Particularly noteworthy is the C-terminal part, which folds differently to those of other group II PLA(2)s. This part is considered to be responsible for inhibition of the platelet-aggregation activity. The calcium-binding loop is tightly organized with sevenfold coordination. Another striking feature of scPLA(2) is the involvement of Asn79 O(delta1) of a symmetry-related molecule in a coordination linkage with Ca(2+) of the calcium-binding loop. This is the first observation of an internal metal ion participating in an intermolecular interaction. The beta-wing of a molecule is deeply inserted into the hydrophobic channel of another molecule and forms several intermolecular interactions. This results in the formation of an infinite chain of molecules. These chains are stacked in an antiparallel arrangement in the crystals.
Amino Acid Sequence↗
Expression, purification, crystallization and preliminary X-ray diffraction studies of recombinant class B non-specific acid phosphatase of Salmonella typhimurium.
The aphA gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The recombinant AphA protein was purified to homogeneity. The protein crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 112.4, b = 130.2, c = 139.6 A. Consistent with the self-rotation function, there are two tetramers in the asymmetric unit, indicating a solvent content of approximately 54%. The crystals are composed of biologically active AphA molecules.
Acid Phosphatase↗
Stone fragility: its therapeutic implications in shock wave lithotripsy of upper urinary tract stones.
OBJECTIVE: To analyse the impact of stone composition on stone fragility (fragmentation) and clearance of upper urinary tract stones after shock wave lithotripsy (SWL). MATERIAL AND METHODS: Between 1st July 1998 and 31st July 2001, 300 renal and ureteric units of 290 patients (10 being bilateral) underwent SWL for upper urinary tract calculi. The degree of fragmentation was divided into four types: (I) Excellent, (II) Good, (III) Fair and (IV) No fragmentation. Stone composition was done by X-ray diffraction crystallography. A statistical comparison was made between degree of fragmentation, number of shock waves delivered, voltage setting, number of sessions required and requirements of adjuvant procedures according to the stone composition. RESULTS: Stone analysis revealed that 90% of the patients had calcium oxalate stones. Of these 80% were calcium oxalate monohydrate (COM) and 20% calcium oxalate dehydrate (COD). Struvite, apatite and uric acid stones comprised of 6%, 3% and 1% respectively. Type-I fragmentation was achieved up to 63.96%, 50% and 100% in COD, struvite and uric stones respectively as compared to 44.9% and 44.44% for COM and apatite stones. Type-III fragmentation was seen up to 8.79% and 33.3% respectively in COM and apatite as compared to 5.55% or less in other types of the stones suggesting that COM and apatite stones produce larger fragments. The mean number of shock waves, voltage and number of treatments was significantly higher for COM and apatite stones (p value < 0.005) with a stone free rate of only 65-66% and 65-68% respectively at three months (p value < 0.001). Similarly the number of adjuvant procedures required in COM alone was more, i.e. 31 as compared to 17 procedures in rest of the other kinds of stones (p value < 0.05). CONCLUSION: Stone composition in Indian subcontinent is different from the western world. Fragility of a stone varies with the composition of the stone and affects the therapeutic results.
Adolescent↗
Design of peptides with branched beta-carbon dehydro-residues: syntheses, crystal structures and molecular conformations of two peptides, (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3.
Highly specific structures can be designed by inserting dehydro-residues into peptide sequences. The conformational preferences of branched beta-carbon residues are known to be different from other residues. As an implication it was expected that the branched beta-carbon dehydro-residues would also induce different conformations when substituted in peptides. So far, the design of peptides with branched beta-carbon dehydro-residues at (i + 1) position has not been reported. It may be recalled that the nonbranched beta-carbon residues induced beta-turn II conformation when placed at (i + 2) position while branched beta-carbon residues induced beta-turn III conformation. However, the conformation of a peptide with a nonbranched beta-carbon residue when placed at (i + 1) position was not found to be unique as it depended on the stereochemical nature of its neighbouring residues. Therefore, in order to induce predictably unique structures with dehydro-residues at (i + 1) position, we have introduced branched beta-carbon dehydro-residues instead of nonbranched beta-carbon residues and synthesized two peptides: (I) N-Carbobenzoxy-DeltaVal-Ala-Leu-OCH3 and (II) N-Carbobenzoxy-DeltaIle-Ala-Leu-OCH3 with DeltaVal and DeltaIle, respectively. The crystal structures of peptides (I) and (II) have been determined and refined to R-factors of 0.065 and 0.063, respectively. The structures of both peptides were essentially similar. Both peptides adopted type II beta-turn conformations with torsion angles; (I): phi1 = -38.7 (4) degrees, psi1 = 126.0 (3) degrees; phi2 = 91.6 (3) degrees, psi2 = -9.5 (4) degrees and (II): phi1 = -37.0 (6) degrees, psi1 = 123.6 (4) degrees, phi2 = 93.4 (4), psi2 = -11.0(4) degrees respectively. Both peptide structures were stabilized by intramolecular 4-->1 hydrogen bonds. The molecular packing in both crystal structures were stabilized in each by two identical hydrogen bonds N1...O1' (-x, y + 1/2, -z) and N2...O2' (-x + 1, y + 1/2, -z) and van der Waals interactions.
Crystallography, X-Ray↗
Design of specific peptide inhibitors of phospholipase A2: structure of a complex formed between Russell's viper phospholipase A2 and a designed peptide Leu-Ala-Ile-Tyr-Ser (LAIYS).
Phospholipase A(2) (EC 3.1.1.4) is a key enzyme of the cascade mechanism involved in the production of proinflammatory compounds known as eicosanoids. The binding of phospholipase A(2) to membrane surfaces and the hydrolysis of phospholipids are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. In order to regulate the production of proinflammatory compounds, a specific peptide inhibitor of PLA(2), Leu-Ala-Ile-Tyr-Ser, has been designed. Phospholipase A(2) from Daboia russelli pulchella (DPLA(2)) and peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) have been co-crystallized. The structure of the complex has been determined and refined to 2.0 A resolution. The structure contains two crystallographically independent molecules of DPLA(2), with one molecule of peptide specifically bound to one of them. The overall conformations of the two molecules are essentially similar except in three regions; namely, the calcium-binding loop including Trp31 (residues 25-34), the beta-wing consisting of two antiparallel beta-strands (residues 74-85) and the C-terminal region (residues 119-133). Of these, the most striking difference pertains to the orientation of Trp31 in the two molecules. The conformation of Trp31 in molecule A was suitable to allow the binding of peptide LAIYS, while that in molecule B prevented the entry of the ligand into the hydrophobic channel. The structure of the complex clearly showed that the OH group of Tyr of the inhibitor formed hydrogen bonds with both His48 N(delta1) and Asp49 O(delta1), while O(gamma)H of Ser was involved in a hydrogen bond with Trp31. Other peptide backbone atoms interact with protein through water molecules, while Leu, Ala and Ile form strong hydrophobic interactions with the residues of the hydrophobic channel.
Amino Acid Sequence↗
Positron emission tomography myocardial perfusion imaging in children with suspected coronary abnormalities.
Positron emission tomography (PET) myocardial perfusion imaging has higher spatial resolution than conventional single photon emission computed tomography (SPECT) imaging and allows accurate and reproducible quantification of myocardial blood flow (MBF). In this article, we describe the role of PET myocardial perfusion imaging in clinical decision making in children with suspected coronary abnormalities. We performed a PET myocardial perfusion study using N-13 ammonia in 10 children (median age, 14 years; range, 1-17 years). The indications included exercise-induced chest pain and ST segment changes during exercise testing, coronary artery ectasia, hypertrophic cardiomyopathy with myocardial bridging of the left anterior descending coronary artery, and suspected left coronary stenosis in an infant with William's syndrome. MBF was assessed at baseline and during adenosine hyperemia in all 10 patients and postexercise in 8 patients. Myocardial perfusion was homogeneous at baseline in all 10 patients, during adenosine perfusion in 9 of 10 patients, and postexercise in all 8 patients. Three patients with homogeneous rest and stress perfusion had impaired myocardial flow reserve. The infant with William's syndrome developed a large, reversible perfusion defect in the left coronary territory during adenosine stress and underwent surgical repair. Myocardial flow reserve findings were valuable for clinical decision making in individual patients. We conclude that MBF quantification with N-13 ammonia and PET provides supplemental perfusion information and is helpful in clinical decision making in children with suspected coronary abnormalities.
Adenosine↗
Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure.
This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).
Animals↗