Structural insights and binding of a natural ligand, succinic acid with serine and cysteine proteases.

In the age of growing infectious diseases, there is a great demand for new inhibitors which can exhibit minimum side effects. Owing to the importance of proteases in life cycle and invasion, they have been projected as attractive targets for structure based drug designing against microbes including viruses. Here we report the inhibitory activity of a well known natural compound succinic acid against both serine and cysteine proteases. The ligand is found co-crystallized with Bovine pancreatic trypsin in one of our crystallization trials and the diffraction data up to1.9 Å reveal its interactions with the catalytic triad residues Histidine 57 and Serine 195. Binding of the ligand with these proteases have been validated using caseinolysis inhibition. With trypsin, ITC analysis showed tight binding of the ligand, resulting in change in Gibb's free energy (ΔG) by -20.31 kJ/mol. To understand the existence of succinic acid at the active site, molecular docking was performed and it revealed binding of it with trypsin and papain at corresponding active sites. This dual inhibitory activity of natural ligand, succinic acid can be accounted for the recent reports on anti-viral property of plant extracts where dicarboxilic fatty acids are normally abundant.

Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases.

The aspartic protease inhibitory efficiency of rBm-33, an aspin from a filarial parasite Brugia malayi was investigated. rBm-33 was found to be thermostable up to 90°C and it forms a stable 'enzyme-product' complex with human pepsin. Aspartic protease inhibitory activity was investigated using UV spectroscopy and isothermal titration calorimetry. Our results suggest that rBm-33 inhibits the activity of important human aspartic proteases that were examined with binding constants (Kb) values between 10.23 × 10(3) and 6.52 × 10(3) M(-1). The binding reactions were enthalpy driven with ΔHb values between -50.99 and -46.07 kJ mol(-1). From kinetic studies, pepsin inhibition by rBm-33 was found to be linear competitive with an inhibition constant (Ki) of 2.5 (±0.8) nM. Because of the inhibitory efficacy of Bm-33 against important human aspartic proteases which play a vital role in immune-regulation along with other functions, Bm-33 can be projected as a drug target for the filariasis.

Expression, purification and characterization of refolded rBm-33 (pepsin inhibitor homolog) from Brugia malayi: a human Lymphatic Filarial parasite.

Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.

Metal induced conformational changes in human insulin: crystal structures of Sr2+, Ni2+ and Cu2+ complexes of human insulin.

Crystal structures of Sr(2+), Ni(2+) and Cu(2+) of human insulin complexes have been determined. The structures of Sr(2+) and Ni(2+) complexes are similar to Zn(2+) insulin and are in T6 conformation. (All the six monomers in the insulin hexamer are in Tensed conformation (T), which means the first eight residues of B-chain are in an extended conformation). Cu(2+) complex, though it assumes T6 conformation, has more structural differences due to lowering of crystal symmetry and space group shift from H3 (Hexagonal crystal system) to P3 (Trigonal crystal system) and a doubling of the c axis. 2Ni(2+) human insulin when compared to 4Ni(2+) Arg insulin suggests that terminal modifications may be responsible for additional metal binding. All the three metals have been shown to have a role in diabetes and hence may be therapeutically useful.