A predominant role for hydrogen bonding in the stability of the homodimer of bothropstoxin-I, A lysine 49-phospholipase A2.
Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-phospholipase A(2) isolated from Bothrops jararacussu venom which damages liposome membranes via a Ca(2+)-independent mechanism. The Glu12/Trp77/Lys80 triad at the dimer interface forms extensive intermolecular hydrogen bonds and hydrophobic contacts, and equilibrium chemical denaturation was used to evaluate the effect on homodimer stability of site-directed mutagenesis of these residues. Changes in the intrinsic fluorescence anisotropy and farUV circular dichroism signals were analyzed using a two-step unfolding model of the BthTx-I dimer to estimate the Gibbs free energy changes of transitions between the dimer and native monomer and between the native and denatured monomers. Whereas the Trp77His, Trp77Gln and Glu12Gln mutants showed native-like dimer stabilities, the Trp77Phe, Lys80Met and Lys80Gly mutants showed significantly reduced K(d) values. A reduced dimer stability is correlated with a decrease in the Ca(2+)-independent membrane damaging activity as monitored by the release of a liposome entrapped fluorescent marker. Although the membrane damaging activity of the monomer is fivefold less than the dimer, the myotoxic activity was unaffected, indicating that these two effects are not correlated. These data suggest that the BthTx-I dimer is predominantly stabilized by hydrogen bonding interactions, and highlight the importance of the homodimeric form for efficient Ca(2+)-independent membrane damage.
Ruller
R
R
Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, FMRP-USP, Ribeirão Preto, SP, Brazil.
Aragão
E A
EA
Chioato
L
L
Ferreira
T Lopes
TL
de Oliveira
A H C
AH
Sà
J M
JM
Ward
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003435:Crotalid Venoms / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D005454:Fluorescence Polarization; D006860:Hydrogen Bonding; D008081:Liposomes / Q000737:chemistry; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry*; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary
D003435:Crotalid Venoms; D008081:Liposomes; C078097:bothropstoxin; D010741:Phospholipases A