Insights on calcium-independent phospholipid membrane damage by Lys49-PLA2 using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu.
Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-PLA2 from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+-independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane.
Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
Tatiana Lopes
TL
Ferreira
Roberto
R
Ruller
Lucimara
L
Chioato
Richard J
RJ
Ward
D000818:Animals; D016880:Anisotropy; D017837:Bothrops / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D054467:Phospholipases A2 / Q000235:genetics / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D003435:Crotalid Venoms; D008081:Liposomes; D010743:Phospholipids; C078097:bothropstoxin; D014364:Tryptophan; D054467:Phospholipases A2; D008239:Lysine; D002118:Calcium