Rajendra K. Singh

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.

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.

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.

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 A2s (PLA2s) 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 PLA2. It was shown earlier that the peptide bound to group II PLA2 specifically and had a dissociation constant (Kd) of 8.8 × 10− 9 M. In the present studies for the binding of LAIYS with a group I PLA2 from Naja naja sagittifera using surface plasmon resonance the dissociation constant was found to be 4.5 × 10− 5 M which is considerably lower than the value found for the group II PLA2. In order to determine the details of binding at the molecular level, a group I PLA2 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 PLA2 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 PLA2. Apparently, the location of Trp 19 in group I PLA2 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 PLA2 it was Tyr (P) OH that formed the corresponding interactions. Tyr (P) in group I PLA2 is the main contributor of the hydrophobic interactions whereas in the complex of LAIYS with group II PLA2 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 PLA2 while a significant portion of the peptide LAIYS was hanging outside the surface of the group I PLA2. The buried area in the complex with group II PLA2 was 811 Å2 whereas, the corresponding area in group I PLA2 was 449 Å2. This shows that the peptide LAIYS is very compatible with the substrate-binding site of group II PLA2 and rather poorly fits into the substrate-binding site of group I PLA2. This indicates that a highly specific ligand for one form of PLA2 may be a poor partner for another form of enzyme.

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 A2 enzymes while the conformation of the corresponding loop in the calcium free monomer deviates considerably with the formation of a unique intraloop Gly33 (N)uf8ffCys27 (O) = 2.74 Å 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 2005.

Comparison of E test and agar dilution for testing activity of ceftriaxone against Neisseria gonorrhoeae.

The Centers for Disease Control and Prevention’s sexually transmitted disease (STD) treatment guidelines, 2015, recommend combination therapy comprising ceftriaxone (250mg) and azithromycin (1 g) for management of gonorrhoea (CDC, 2015). The recent data from WHO SEAR (South-East Asia region) countries attest to the effectiveness of in-use therapies. Further, no immediate threat to the efficacy of the extendedspectrum cephalosporins (ESCs) is suggested (Bala et al., 2013). However, in view of the reports of creeping MICs to ESCs world over (Martin et al., 2006; Unemo et al., 2010; Ison et al., 2011; Ohnishi et al., 2011; Sood et al., 2013; Lahra et al., 2014), we can now envisage that gonococcus is slowly walking towards resistance. Therefore, there is a need to keep a close watch on changing susceptibility to ESCs. We are well aware that the disc-diffusion method does not detect small changes in antimicrobial susceptibilities and that these are reflected only by methods producing quantitative MIC results. E test is a very convenient method for MIC determination in a clinical bacteriology laboratory and is widely used. We therefore sought to determine how closely E test results correlate to those of the agar dilution method, the reference ‘gold standard’ for MIC determination of ceftriaxone in Neisseria gonorrhoeae.

Structure of the zinc-induced heterodimer of two calcium-free isoforms of phospholipase A2 from Naja naja sagittifera at 2.7 Å resolution

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.

Exploring quinolone resistance-determining region in Neisseria gonorrhoeae isolates from across India

Background & objectives: Antimicrobial resistance in Neisseria gonorrhoeae, the causative agent of gonorrhoea, is a subject of worldwide attention. The present study was undertaken to examine the rates of ciprofloxacin resistance, to correlate mutations in gyrA and parC genes with the level of resistance and to look for a variation in mutation pattern, if any, in isolates from across the country. Methods: A total of 113 isolates of N. gonorrhoeae collected from sexually transmitted infection patients in six centres during November 2010 to October 2013 were investigated. Minimum inhibitory concentration (MIC) determination was done by E-test and results interpreted as per Calibrated Dichotomous Sensitivity criteria. DNA sequence analysis of gyrA and parC genes was done. Results: Of the 113 isolates, only three (2.6%) were susceptible whereas eight (7.07%) were less susceptible, 32 [28.3%, 95% confidence interval (CI): 20.4-37.6%] resistant (MIC 1-3 µg/ml) and 70 (61.9%, 95% CI: 52.2-70.7%) exhibited high-level resistance (HLR) (MIC ≥4 µg/ml) to ciprofloxacin. A S91F substitution in gyrA gene was demonstrated in all ciprofloxacin non-susceptible isolates. All resistant and HLR isolates had a double mutation in gyrA gene. However, only 5.7 per cent of HLR isolates showed double mutations in parC gene. One isolate (MIC 32 µg/ml) had a previously undescribed G85D substitution in the parC gene. Interpretation & conclusions: A S91F substitution in gyrA gene was seen in all non-susceptible isolates of N. gonorrhoeae. It may be used as a marker for ciprofloxacin resistance for molecular surveillance approaches to complement the culture-based methods.