Characterization of suramin binding sites on the human group IIA secreted phospholipase A2 by site-directed mutagenesis and molecular dynamics simulation.

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change.

Active site mutants of human secreted Group IIA Phospholipase A2 lacking hydrolytic activity retain their bactericidal effect.

The Human Secreted Group IIA Phospholipase A(2) (hsPLA2GIIA) presents potent bactericidal activity, and is considered to contribute to the acute-phase immune response. Hydrolysis of inner membrane phospholipids is suggested to underlie the bactericidal activity, and we have evaluated this proposal by comparing catalytic activity with bactericidal and liposome membrane damaging effects of the G30S, H48Q and D49K hsPLA2GIIA mutants. All mutants showed severely impaired hydrolytic activities against mixed DOPC:DOPG liposome membranes, however the bactericidal effect against Micrococcus luteus was less affected, with 50% killing at concentrations of 1, 3, 7 and 9 μg/mL for the wild-type, D49K, H48Q and G30S mutants respectively.

Shared structural determinants for the calcium-independent liposome membrane permeabilization and sarcolemma depolarization in Bothropstoxin-I, a LYS49-PLA(2) from the venom of Bothrops jararacussu.

The structural determinants of myotoxicity of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A(2) from Bothrops jararacussu venom, were studied by measuring the resting membrane potential in the mouse phrenic nerve-diaphragm preparation. This method proved to be around 100-fold more sensitive than the creatine kinase release assay, and was used to evaluate a total of 31 site-directed BthTX-I alanine scanning mutants. Mutants that reduced the resting membrane potential were located in a surface patch defined by residues in the C-terminal loop (residues 115-129), positions 37-39 in the membrane interfacial recognition surface (Y46 and K54), and residue K93.

Mapping of suramin binding sites on the group IIA human secreted phospholipase A2.

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein, namely the active-site and residues located in the N- and C-termini, respectively.

Permeabilization of E. coli K12 inner and outer membranes by bothropstoxin-I, A LYS49 phospholipase A2 from Bothrops jararacussu.

Although lacking catalytic activity, the Lys49-PLA(2)s damage artificial membranes by a Ca(2+)-independent mechanism, and demonstrate a potent bactericidal effect. The relationship between the membrane-damaging activity and bactericidal effect of bothropstoxin-I (BthTx-I), a Lys49-PLA(2) from the venom of Bothrops jararacussu, was evaluated for the wild-type protein and a series of site-directed mutants in the active site and C-terminal regions of the protein. The membrane permeabilization effect against the inner and outer membranes of Escherichia coli K12 was evaluated by fluorescence changes of Sytox Green and N-phenyl-N-naphthylamine, respectively.

Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis.

Scanning alanine mutagenesis has been used to study the structural determinants of several activities of bothropstoxin-I (BthTx-I), a lysine 49 Phospholipases A(2) from the venom of Bothrops jararacussu. A total of 31 mutants were generated in the interfacial recognition site and C-terminal loop regions of the protein. The effects of mutagenesis on the in vivo myotoxic activity, the cytolytic activity against cultured C2C12 myoblasts, the bactericidal activity, and the Ca(2+)-independent membrane damaging activity against liposome membranes were compared.