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.
α-type phospholipase A2 inhibitory protein (PLIα) isolated from the serum of the venomous snake Glyoidius brevicaudus, GbPLIα, is a homotrimer of subunits having a C-type lectin-like domain. The serum protein from nonvenomous snake Elaphe quadrivirgata, EqPLIα-LP, is homologous to GbPLIα, but it does not show any inhibitory activity against PLA2s. When a mixture of denaturant-treated monomeric forms of GbPLIα and EqPLIα-LP was used to reconstitute their trimers, no significant amounts of heterotrimers composed of GbPLIα and EqPLIα-LP subunits could be formed. On the other hand, when a mixture of denaturant-treated monomeric forms of GbPLIα and the recombinant chimeric EqPLIα-LP, Eq13Gb37Eq, in which the residues 13-36 were replaced by those of GbPLIα, was used to reconstitute their trimers, significant amounts of their heterotrimers were observed. Furthermore, when a mixture of denaturant-treated monomeric forms of EqPLIα-LP and the recombinant chimeric GbPLIα, Gb13Eq37Gb, in which the residues 13-36 were replaced by those of EqPLIα-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 GbPLIα 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 GbPLIα with a single amino acid substitution, GbPLIα(K28E).
Nishida
Masanori
M
Laboratory of Biochemistry, Osaka University of Pharmaceutical Sciences, Osaka, Japan.
Okamoto
Masataka
M
Ohno
Ai
A
Okumura
Kohji
K
Hayashi
Kyozo
K
Ikeda
Kiyoshi
K
Inoue
Seiji
S
D000818:Animals; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D054513:Group IV Phospholipases A2 / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D054467:Phospholipases A2 / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012911:Snakes; D055672:Static Electricity; D020349:Surface Plasmon Resonance
D011994:Recombinant Proteins; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D019791:Guanidine