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

Abstract

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.

Figure 1. Far-UV CD spectra of native and recombinant BthTx-I after refolding and purification

(A) The native BthTx-I purified from B. jararacussu venom (▪), the wild-type recombinant BthTx-I (□) and the mutants L106F (♦) and L5F/V102A/L106F (▵). (B) Wild-type recombinant BthTx-I (thick solid line, □) and the mutants L106A (▵) and L5F/L106F (▴). (C) Wild-type recombinant BthTx-I (thick solid line, □) and the mutants L5F (•), V102A (▴), L5F/V102A (○) and V102A/L106F (▵). (D) Ratio of the observed ellipticity signals at 222 and 209 nm for native BthTx-I (Nat), wild-type recombinant BthTx-I (Rec) and substrate-binding region mutants.

Figure 2. Fraction of unfolded protein f_u as a function of temperature for BthTx-I and substrate-binding region mutants

Native BthTx-I (□), wild-type recombinant BthTx-I (♦) and the mutants L5F (▾), V102A (•), L106F (○), L5F/V102A (crossed diamonds), V102A/L106F (+) and L5F/V102A/L106F (▵). The inset presents the values of melting temperature T_m of BthTx-I before and after site-directed mutagenesis of residues in the substrate-binding region; n.d., value not determined.

Figure 3. Plasma levels of CK activity 3 h after injection of 30 μg of native BthTx-I (Nat), wild-type recombinant BthTx-I (Rec) and substrate-binding site mutants into the gastrocnemius muscle of mice

The control experiment used an identical volume of PBS in the absence of protein. Results are expressed as the means±S.D. for a minimum of five independent experiments.

Figure 4. Release of entrapped calcein from liposomes composed of EYPC/DMPA at a 9:1 molar ratio 150 s after mixing with native BthTx-I (Nat), wild-type recombinant (Rec) and substrate-binding-site mutants at a protein/lipid ratio of 1:400

The final protein concentration in all experiments was 3 μg·ml⁻¹, and the results are expressed as mean percentage of total calcein release after the addition of 5 mM Triton X-100.

Figure 5. Transparent surface representation of the substrate-binding regions of (A) BthTx-I and (B) human group II secreted PLA₂ (PDB code 1BBC; [35]), showing the active-site (positions 48, 49, 52 and 99) and substrate-binding residues (positions 5, 22, 102 and 106) for both proteins

The solvent-accessible surface is shown, and the dark grey area in each Figure corresponds to the exposed surfaces of the side chains at positions 5, 102 and 106. The Figure was prepared using SwissPDB software [39].