High metal substitution tolerance of anthrax lethal factor and characterization of its active copper-substituted analogue

Anthrax lethal factor (LF) is a zinc-dependent metalloendopeptidase and a member of the gluzincin family. The current report demonstrates a high metal substitution tolerance of LF atypical of gluzincins and other zinc-dependent metalloproteases. Mn2 +, Co2 +, Ni2 +, Cu2 + and Cd2 + were found to reactivate the apoprotein of LF to a level either comparable to or significantly higher than that noted for the native zinc enzyme. The most active form of LF was obtained with Cu2 +, a surprising observation since most Cu2 +-substituted zinc proteases display very low activity. Cu2 +-substituted LF (CuLF), prepared by direct exchange and by apoprotein reconstitution methodologies, displayed a several-fold higher catalytic competence towards chromogenic and fluorogenic LF substrates than native LF. CuLF bound Cu2 + tightly with a dissociation constant in the femtomolar range. The electron paramagnetic resonance spectrum of CuLF revealed the protein-bound metal ion to be coordinated to two nitrogen donor atoms, suggesting that Cu2 + binds to both active site histidine residues. While ZnLF and CuLF (prepared by direct exchange) were capable of killing RAW 264.7 murine macrophage-like cells, apoLF and all metal-reconstituted apoprotein preparations failed to elicit a cytotoxic response. Competition experiments using apoLF/ZnLF mixtures demonstrated the propensity of apoLF to relieve ZnLF-induced cell death, suggesting that both protein forms can compete with each other for binding to protective antigen. The lack of cytotoxicity of apoLF and its metal-reconstituted variants likely originates from structural perturbations in these proteins which might prevent their translocation into the cytoplasm.

Graphical abstractAnthrax lethal factor (LF) is a zinc-dependent metalloprotease. This study shows that LF is active with a variety of other metals, particularly with copper, which increases the catalytic competence of the enzyme several-fold.Figure optionsDownload full-size imageDownload as PowerPoint slide