Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity

摘要

BthTx-I (bothropstoxin-I) is a myotoxic Lys⁴⁹-PLA2 (phospholipase A2 with Lys⁴⁹) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca²⁺-independent mechanism. The highly conserved Phe⁵/Ala¹⁰²/Phe¹⁰⁶ motif in the hydrophobic substrate-binding site of the Asp⁴⁹-PLA2s is substituted by Leu⁵/Val¹⁰²/Leu¹⁰⁶ in the Lys⁴⁹-PLA2s. The Leu⁵/Val¹⁰²/Leu¹⁰⁶ triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe⁵/Ala¹⁰²/Phe¹⁰⁶ mutant. All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca²⁺-independent membrane-damaging activities of the recombinant proteins, were evaluated. The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu¹⁰⁶→Phe) and L5F/F102A/L106F mutant proteins were identical. The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm. Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker. However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity. This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca²⁺-independent membrane damage, and implies that substrate binding retains an important role in this process.