[Show abstract][Hide abstract] ABSTRACT: Resistance against currently used antitubercular therapeutics increasingly undermines efforts to contain the worldwide tuberculosis (TB) epidemic. Recently, benzothiazinone (BTZ) inhibitors have shown nanomolar potency against both drug-susceptible and multidrug-resistant strains of the tubercle bacillus. However, their proposed mode of action is lacking structural evidence. We report here the crystal structure of the BTZ target, FAD-containing oxidoreductase Mycobacterium tuberculosis DprE1, which is essential for viability. Different crystal forms of ligand-free DprE1 reveal considerable levels of structural flexibility of two surface loops that seem to govern accessibility of the active site. Structures of complexes with the BTZ-derived nitroso derivative CT325 reveal the mode of inhibitor binding, which includes a covalent link to conserved Cys387, and reveal a trifluoromethyl group as a second key determinant of interaction with the enzyme. Surprisingly, we find that a noncovalent complex was formed between DprE1 and CT319, which is structurally identical to CT325 except for an inert nitro group replacing the reactive nitroso group. This demonstrates that binding of BTZ-class inhibitors to DprE1 is not strictly dependent on formation of the covalent link to Cys387. On the basis of the structural and activity data, we propose that the complex of DrpE1 bound to CT325 is a representative of the BTZ-target complex. These results mark a significant step forward in the characterization of a key TB drug target.
Proceedings of the National Academy of Sciences 06/2012; 109(28):11354-9. DOI:10.1073/pnas.1205735109 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Galectins are involved in many cellular processes due to their ability to bind carbohydrates. Understanding their functions has shown the necessity for potent and specific galectin inhibitors. Human galectin-7 (hGal-7), in particular, has been highlighted as an important marker in many types of cancer by either inhibiting or promoting tumour growth. Producing ligands able to selectively target hGal-7 will offer promising tools for deciphering cancer processes in which hGal-7 is involved as well as present potential solutions for future therapeutics. Here we report the high resolution crystal structure of hGal-7 in complex with a synthetic 2-O-benzylphosphate-galactoside inhibitor (which is > 60-fold more potent than its parent galactoside). The high resolution crystallographic analysis highlights the validity of using saccharide derivatives, conserving properties of the galactose binding, while enhanced affinity and specificity is provided by the added phosphate group. This structural information will allow the design of further inhibitors with improved potency and specificity.
The atomic coordinates for the complex of human galectin-7 as well as for the free structure have been deposited with the Protein Data Bank (accession numbers 3ZXE and 3ZXF respectively)
Structured digital abstract
hGal-7 and hGal-7
bind by X-raycrystallography (Viewinteraction)
[Show abstract][Hide abstract] ABSTRACT: Long term survival of the pathogen Mycobacterium tuberculosis in humans is linked to the immunomodulatory potential of its complex cell wall glycolipids, which include the phosphatidylinositol mannoside (PIM) series as well as the related lipomannan and lipoarabinomannan glycoconjugates. PIM biosynthesis is initiated by a set of cytosolic α-mannosyltransferases, catalyzing glycosyl transfer from the activated saccharide donor GDP-α-D-mannopyranose to the acceptor phosphatidyl-myo-inositol (PI) in an ordered and regio-specific fashion. Herein, we report the crystal structure of mannosyltransferase Corynebacterium glutamicum PimB' in complex with nucleotide to a resolution of 2.0 Å. PimB' attaches mannosyl selectively to the 6-OH of the inositol moiety of PI. Two crystal forms and GDP- versus GDP-α-d-mannopyranose-bound complexes reveal flexibility of the nucleotide conformation as well as of the structural framework of the active site. Structural comparison, docking of the saccharide acceptor, and site-directed mutagenesis pin regio-selectivity to a conserved Asp residue in the N-terminal domain that forces presentation of the correct inositol hydroxyl to the saccharide donor.
[Show abstract][Hide abstract] ABSTRACT: Long term survival of the pathogen Mycobacterium tuberculosis in humans is linked to the immunomodulatory potential of its complex cell wall glycolipids, which include the phosphatidylinositol
mannoside (PIM) series as well as the related lipomannan and lipoarabinomannan glycoconjugates. PIM biosynthesis is initiated
by a set of cytosolic α-mannosyltransferases, catalyzing glycosyl transfer from the activated saccharide donor GDP-α-d-mannopyranose to the acceptor phosphatidyl-myo-inositol (PI) in an ordered and regio-specific fashion. Herein, we report the crystal structure of mannosyltransferase Corynebacterium glutamicum PimB′ in complex with nucleotide to a resolution of 2.0 Å. PimB′ attaches mannosyl selectively to the 6-OH of the inositol
moiety of PI. Two crystal forms and GDP- versus GDP-α-d-mannopyranose-bound complexes reveal flexibility of the nucleotide conformation as well as of the structural framework of
the active site. Structural comparison, docking of the saccharide acceptor, and site-directed mutagenesis pin regio-selectivity
to a conserved Asp residue in the N-terminal domain that forces presentation of the correct inositol hydroxyl to the saccharide
Journal of Biological Chemistry 11/2010; 285(48):37741-37752. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) B effects blood vessel formation by binding to VEGF receptor 1. To study the specifics of the biological profile of VEGF-B in both physiological and pathological angiogenesis, a neutralising anti-VEGF-B antibody (2H10) that functions by inhibiting the binding of VEGF-B to VEGF receptor 1 was developed. Here, we present the structural features of the ‘highly ordered’ interaction of the Fab fragment of this antibody (Fab-2H10) with VEGF-B. Two molecules of Fab-2H10 bind to symmetrical binding sites located at each pole of the VEGF-B homodimer, giving a unique U-shaped topology to the complex that has not been previously observed in the VEGF family. VEGF-B residues essential for binding to the antibody are contributed by both monomers of the cytokine. Our detailed analysis reveals that the neutralising effect of the antibody occurs by virtue of the steric hindrance of the receptor-binding interface. These findings suggest that functional complementarity between VEGF-B and 2H10 can be harnessed both in analysing the therapeutic potential of VEGF-B and as an antagonist of receptor activation.
[Show abstract][Hide abstract] ABSTRACT: Interferon-gamma-inducible protein (IP-10) belongs to the CXC class of chemokines and plays a significant role in the pathophysiology of various immune and inflammatory responses. It is also a potent angiostatic factor with antifibrotic properties. The biological activities of IP-10 are exerted by interactions with the G-protein-coupled receptor CXCR3 expressed on Th1 lymphocytes. IP-10 thus forms an attractive target for structure-based rational drug design of anti-inflammatory molecules. The crystal structure of mouse IP-10 has been determined and reveals a novel tetrameric association. In the tetramer, two conventional CXC chemokine dimers are associated through their N-terminal regions to form a 12-stranded elongated beta-sheet of approximately 90 A in length. This association differs significantly from the previously studied tetramers of human IP-10, platelet factor 4 and neutrophil-activating peptide-2. In addition, heparin- and receptor-binding residues were mapped on the surface of IP-10 tetramer. Two heparin-binding sites were observed on the surface and were present at the interface of each of the two beta-sheet dimers. The structure supports the formation of higher order oligomers of IP-10, as observed in recent in vivo studies with mouse IP-10, which will have functional relevance.
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2 Ǻ resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other α-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3 Å for their Cα tracings. The overall fold of the toxin is very similar to other scorpion α-toxins. It is a βαββ protein. The β-sheet involves residues Glu2-Ile6 (strand β1), Asp32-Trp39 (strand β3) and Val45-Val55 (strand β4). The single α-helix formed is by residues Asn19-Asp28 (α2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical α-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.
[Show abstract][Hide abstract] ABSTRACT: Secretory low molecular weight phospholipase A(2)s (PLA(2)s) are believed to be involved in the release of arachidonic acid, a precursor for the biosynthesis of pro-inflammatory eicosanoids. Therefore, the specific inhibitors of these enzymes may act as potent anti-inflammatory agents. Similarly, the compounds with known anti-inflammatory properties should act as specific inhibitors. Two plant compounds, (a) anisic acid (4-methoxy benzoic acid) and (b) atropine (8-methyl-8-azabicyclo oct-3-hydroxy-2-phenylpropanoate), have been used in various inflammatory disorders. Both compounds (a) and (b) have been found to inhibit PLA(2) activity having binding constants of 4.5 x 10(-5) M and 2.1 x 10(-8) M, respectively. A group IIA PLA(2) was isolated and purified from the venom of Daboia russelli pulchella (DRP) and its complexes were made with anisic acid and atropine. The crystal structures of the two complexes (i) and (ii) of PLA(2) with compounds (a) and (b) have been determined at 1.3 and 1.2 A resolutions, respectively. The high-quality observed electron densities for the two compounds allowed the accurate determinations of their atomic positions. The structures revealed that these compounds bound to the enzyme at the substrate - binding cleft and their positions were stabilized by networks of hydrogen bonds and hydrophobic interactions. The most characteristic interactions involving Asp 49 and His 48 were clearly observed in both complexes, although the residues that formed hydrophobic interactions with these compounds were not identical because their positions did not exactly superimpose in the large substrate-binding hydrophobic channel. Owing to a relatively small size, the structure of anisic acid did not alter upon binding to PLA(2), while that of atropine changed significantly when compared with its native crystal structure. The conformation of the protein also did not show notable changes upon the bindings of these ligands. The mode of binding of anisic acid to the present group II PLA(2) is almost identical to its binding with bovine pancreatic PLA(2) of group I. On the other hand, the binding of atropine to PLA(2) is similar to that of another plant alkaloid aristolochic acid.
Proteins Structure Function and Bioinformatics 07/2006; 64(1):89-100. DOI:10.1002/prot.20970 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Phospholipase A(2) (PLA(2); EC 18.104.22.168) catalyzes the first step of the production of proinflammatory compounds collectively known as eicosanoids. The binding of phospholipid substrates to PLA(2) occurs through a well formed hydrophobic channel. Surface plasmon resonance studies have shown that niflumic acid binds to Naja naja sagittifera PLA(2) with an affinity that corresponds to a dissociation constant (K(d)) of 4.3 x 10(-5) M. Binding studies of PLA(2) with niflumic acid were also carried out using a standard PLA(2) kit that gave an approximate binding constant, K(i), of 1.26 +/- 0.05 x 10(-6) M. Therefore, in order to establish the viability of PLA(2) as a potential target molecule for drug design against inflammation, arthritis and rheumatism, the three-dimensional structure of the complex of PLA(2) with the known anti-inflammatory agent niflumic acid [2-[3-(trifluoromethyl)anilino]nicotinic acid] has been determined at 2.5 Angstroms resolution. The structure of the complex has been refined to an R factor of 0.187. The structure determination reveals the presence of one niflumic acid molecule at the substrate-binding site of PLA(2). It shows that niflumic acid interacts with the important active-site residues His48 and Asp49 through two water molecules. It is observed that the niflumic acid molecule is completely buried in the substrate-binding hydrophobic channel. The conformations of the binding site in PLA(2) as well as that of niflumic acid are not altered upon binding. However, the orientation of the side chain of Trp19, which is located at the entry of the substrate-binding site, has changed from that found in the native PLA(2), indicating its familiar role.
[Show abstract][Hide abstract] ABSTRACT: Secretory phospholipase A2s (PLA2s), the structurally-homologous enzymes share a common qualitative catalytic site, but differ greatly in their pharmacological properties and toxicities. There has been a recognizable pattern of mutations in the primary sequence of PLA2s that alter their catalytic properties significantly. In the present study, the amino acid sequence and the three-dimensional structure of a new isoform of PLA2 from crude venom of Indian cobra sub-species Naja naja sagittifera (N.n.s.) has been determined by X-ray crystallography. The crystal structure has revealed several novel features of PLA2 folding and function. It contains 913 protein atoms and one each of Ca2+, phosphate and acetate ions with 142 solvent water molecules. A Ca2+ ion is present in the calcium-binding loop and forms a seven-fold coordination with a distorted pentagonal bipyramidal geometry. One of the coordination linkages is with the acetate ion, instead of the conserved water molecule. The presence of Lys at position 31 has a stabilizing effect on the loop Tyr 25-Cys 29 by interacting with carbonyl oxygen atoms of Tyr 25, Gly 26 and Cys 29. In turn, it lends stability to the Ca(2+)-binding loop as well. Another unique feature of the PLA2 structure is the formation of an intrastrand hydrogen bond, involving Ogamma of Thr 73 and Oepsilon2 of Glu 71, thus helping the beta-wing to act as a sharp arrow for insertion into other molecules. Yet another important feature of this PLA2 pertains to the conformation of its C-terminal segment, which is stabilized by a unique hydrogen bond through carbonyl oxygen of Lys 116 and Ndelta2 of Asn 120. This structural feature may be useful in the molecular recognition of the PLA2 through C-terminal segment.
Indian journal of biochemistry & biophysics 10/2005; 42(5):279-86. · 0.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of the zinc-saturated C-terminal lobe of bovine lactoferrin has been determined at 2.0 A resolution using crystals stabilized at pH 3.8. This is the first metal-saturated structure of any functional lactoferrin at such a low pH. Purified samples of proteolytically generated zinc-saturated C-terminal lobe were crystallized from 0.1 M MES buffer pH 6.5 containing 25%(v/v) polyethyleneglycol monomethyl ether 550 and 0.1 M zinc sulfate heptahydrate. The crystals were transferred to 25 mM ammonium acetate buffer containing 25%(v/v) polyethyleneglycol monomethyl ether 550 and the pH was gradually changed from 6.5 to 3.8. The X-ray intensity data were collected with a 345 mm imaging-plate scanner mounted on an RU-300 rotating-anode X-ray generator using crystals soaked in the buffer at pH 3.8. The structure was determined with the molecular-replacement method using the coordinates of the monoferric C-terminal lobe of bovine lactoferrin as a search model and was refined to an R factor of 0.192 for all data to 2.0 A resolution. The final model comprises 2593 protein atoms (residues 342-676 and 681-685), 138 carbohydrate atoms (from 11 monosaccharide units in three glycan chains), three Zn2+ ions, one CO3(2-) ion, one SO(4)2- ions and 227 water molecules. The overall folding of the present structure is essentially similar to that of the monoferric C-terminal lobe of bovine lactoferrin, although it contains Zn2+ in place of Fe3+ in the metal-binding cleft as well as two additional Zn2+ ions on the surface of the C-terminal lobe. The Zn2+ ion in the cleft remains bound to the lobe with octahedral coordination. The bidentate carbonate ion is stabilized by a network of hydrogen bonds to Ala465, Gly466, Thr459 and Arg463. The other two zinc ions also form sixfold coordinations involving symmetry-related protein and water molecules. The number of monosaccharide residues from the three glycan chains of the C-terminal lobe was 11, which is the largest number observed to date. The structure shows that the C-terminal lobe of lactoferrin is capable of sequestering a Zn2+ ion at a pH of 3.8. This implies that the zinc ions can be sequestered over a wide pH range. The glycan chain attached to Asn545 may also have some influence on iron release from the C-terminal lobe.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Drug Targeting 08/2005; 13(6):367-74. DOI:10.1080/10611860500254450 · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Proteins Structure Function and Bioinformatics 06/2005; 59(4):856-63. DOI:10.1002/prot.20464 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of a zinc-induced heterodimer of two metal-free isoforms of a cobra venom phospholipase A(2) has been determined at 2.7 angstroms resolution. The crystals belong to space group P4(1), with unit-cell parameters a = b = 65.5, c = 58.4 angstroms, and have a single dimer in the asymmetric unit. The structure has been refined to R(cryst) and R(free) factors of 0.188 and 0.232, respectively. The two isoforms have a sequence identity of 82%. The zinc ion forms a fivefold coordination with a trigonal bipyramidal geometry involving one O atom each from Asp24 and Asn112 from molecule A and Asp24 from molecule B and two water molecules. Both molecules of the dimer are inactive. Molecule A is inactive because Arg31 (B) binds to Asp49 (A), while an acetate ion has displaced the essential water molecule and interacts with His48 (A). On the other hand, Arg31 (A) interacts with the calcium-binding loop of molecule B, resulting in an altered conformation of the loop. The absence of a calcium ion, loss of the essential water molecule and the altered conformation of the calcium-binding loop may be the reasons for the loss of activity of molecule B.
[Show abstract][Hide abstract] ABSTRACT: Phospholipase A2 is potentially an important target for structure-based rational drug design. In order to determine the involvement of phospholipase A2 in the action of non-steroidal anti-inflammatory drugs (NSAIDs), the crystal structure of the complex formed between phospholipase A2 and aspirin has been determined at 1.9 angstroms resolution. The structure contains 915 protein atoms, 1 calcium ion, 13 atoms of aspirin and 105 water molecules. The observed electron density of the aspirin molecule in the structure was of very high quality thus allowing the precise determination of its atomic coordinates leading to the clear description of its interactions with the enzyme. The structure of the complex clearly shows that aspirin is literally embedded in the hydrophobic environment of PLA2. It is so placed in the substrate binding channel that it forms several important attractive interactions with calcium ion, His 48 and Asp 49. Thus, the structure of the complex clearly shows that aspirin occupies a favourable place in the specific binding site of PLA2. The binding studies have shown that acetyl salicylate (aspirin) binds to PLA2 enzyme specifically with a dissociation constant of 6.4 x 10(-6) M. The structural details and binding data suggest that the inhibition of PLA2 by aspirin is of pharmacological
Journal of Drug Targeting 03/2005; 13(2):113-9. DOI:10.1080/10611860400024078 · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.
Proteins Structure Function and Bioinformatics 02/2005; 62(2):329-37. DOI:10.1002/prot.20708 · 2.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 +/- 0.3 x 10(-11) M and thus works as a highly potent competitive inhibitor. The commercially available alpha-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated alpha-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 M ammonium sulfate. The rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3) were obtained. The X-ray intensity data were collected at 2.2 angstroms resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of alpha-chymotrypsin, which was readily interpreted as a 14 residue fragment of alpha-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 sigma between the active site Ser195 Ogamma and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16-Trp29. In addition to the O-C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of alpha-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of alpha-chymotrypsin.
[Show abstract][Hide abstract] ABSTRACT: Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in α-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 ± 0.3 × 10−11 m and thus works as a highly potent competitive inhibitor. The commercially available α-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated α-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mm sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 m ammonium sulfate. The rectangular crystals of small dimensions of 0.24 × 0.15 × 0.10 mm3 were obtained. The X-ray intensity data were collected at 2.2 Å resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of α-chymotrypsin, which was readily interpreted as a 14 residue fragment of α-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16–29). The electron density for the eight residues of the C-terminus, i.e. Ala22–Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16–Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 σ between the active site Ser195 Oγ and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 Å between Ser195 Oγ and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16–Trp29. In addition to the O–C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of α-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of α-chymotrypsin.