Inhibitory activities of the heterotrimers formed from two α-type phospholipase A2 inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation

alpha-type phospholipase A(2) inhibitory protein (PLI alpha) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLI alpha, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLI alpha-LP, is homologous to GbPLI alpha, but it does not show any inhibitory activity against PLA(2)s. When a mixture of denaturant-treated monomeric forms of GbPLI alpha and EqPLI alpha-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLI alpha and EqPLI alpha-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLI alpha and the recombinant chimeric EqPLI alpha-LP, Eq13Gb37Eq, in which the residues 13-36 were replaced by those of GbPLI alpha, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLI alpha-LP and the recombinant chimeric GbPLI alpha, Gb13Eq37Gb, in which the residues 13-36 were replaced by those of EqPLI alpha-LP, was used, significant amounts of their heterotrimers were observed. By comparison of the respective inhibitory activities of the heterotrimeric subspecies, it was suggested that the inhibitory activity of the trimer was governed by one subunit with the highest activity, and not affected by the number of these subunits. The intermolecular electrostatic interactions between Glu23 and Lys28 of GbPLI alpha were also suggested to be important in stabilizing the trimeric structure. The importance of the electrostatic interaction was supported by the less stability of the homotrimeric structure of a mutant GbPLI alpha with a single amino acid substitution, GbPLI alpha(K28E). (C) 2010 Elsevier B.V. All rights reserved.