Talat Jabeen

Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors.

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.

Aspirin induces its anti-inflammatory effects through its specific binding to phospholipase A2: Crystal structure of the complex formed between phospholipase A2 and aspirin at 1.9 Å resolution

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 Å 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 6.4×10−6 M. The structural details and binding data suggest that the inhibition of PLA2 by aspirin is of pharmacological significance and part of its anti-inflammatory effects may be due to its binding with PLA2.

Crystal structures of the complexes of a group IIA phospholipase A2 with two natural anti-inflammatory agents, anisic acid, and atropine reveal a similar mode of binding

Secretory low molecular weight phospholipase A2s (PLA2s) 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 PLA2 activity having binding constants of 4.5 × 10−5 M and 2.1 × 10−8 M, respectively. A group IIA PLA2 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 PLA2 with compounds (a) and (b) have been determined at 1.3 and 1.2 Å 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 PLA2, 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 PLA2 is almost identical to its binding with bovine pancreatic PLA2 of group I. On the other hand, the binding of atropine to PLA2 is similar to that of another plant alkaloid aristolochic acid. Proteins 2006.

Acceptor substrate discrimination in phosphatidyl-myo-inositol mannoside synthesis: structural and mutational analysis of mannosyltransferase Corynebacterium glutamicum PimB'.

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.

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.

Structure of the zinc-saturated C-terminal lobe of bovine lactoferrin at 2.0 A resolution.

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.

Non-steroidal anti-inflammatory drugs as potent inhibitors of phospholipase A2: structure of the complex of phospholipase A2 with niflumic acid at 2.5 Å resolution

Phospholipase A(2) (PLA(2); EC 3.1.3.4) 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.

Structural studies on the cobra venom factor: isolation, purification, crystallization and preliminary crystallographic analysis

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. It is a three-chain glycoprotein with a molecular weight of 149,000 Da. In serum, CVF forms a bimolecular enzyme with the Bb subunit of factor B. The enzyme cleaves C3 and C5, causing complement consumption in human and mammalian serum. CVF is frequently used to decomplement serum to investigate the biological functions of complement and serves as a tool to investigate the multifunctionality of C3. Furthermore, CVF bears the potential for clinical application to deplete complement in situations where complement activation is involved in the pathogenesis of disease. CVF was isolated from Indian cobra (Naja naja naja) venom. The protein was crystallized at room temperature using the sitting-drop vapour-diffusion technique. The crystals diffract to 2.7 A resolution and belong to the tetragonal space group P4(1), with unit-cell parameters a = b = 62.7, c = 368.1 A.

Detection of native peptides as potent inhibitors of enzymes

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.

Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 A resolution reveals the presence of a new PLA2-like protein with a novel cys 32-Cys 49 disulphide bridge with a bound sugar at the substrate-binding site

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 importantance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 Å and c = 68.4 Å 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 2006.