Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2.
Bothropstoxin-I (BthTx-I) is a Lys(49)-phospholipase A(2) from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca(2+)-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117-122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys(122), these same substitutions did not significantly alter the Ca(2+)-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca(2+)-independent membrane-damaging activity but, with the exception of Lys(122), had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115-119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A(2), and support a model of a Ca(2+)-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.
Chioato
Lucimara
L
Departamento de Bioqui;mica e Imunologia, FMRP-USP, Universidade de São Paulo, Brazil.
De Oliveira
Arthur H C
AH
Ruller
Roberto
R
Sá
Juliana M
JM
Ward
Richard J
RJ
D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D017837:Bothrops / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D008051:Lipid Bilayers; D008081:Liposomes; D011994:Recombinant Proteins; D010741:Phospholipases A; D008239:Lysine; D000409:Alanine; D002118:Calcium