title: High metal substitution tolerance of anthrax lethal factor and characterization of its active copper-substituted analogue.
abstract: 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. Mn(2+), Co(2+), Ni(2+), Cu(2+) and Cd(2+) 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 Cu(2+), a surprising observation since most Cu(2+)-substituted zinc proteases display very low activity. Cu(2+)-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 Cu(2+) 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 Cu(2+) 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.
authors.affiliation: Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON P3E 2C6, Canada.
authors.forename: Suet Y
authors.initials: SY
authors.lastname: Lo
authors.affiliation: Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON P3E 2C6, Canada.
authors.forename: Crystal E
authors.initials: CE
authors.lastname: Säbel
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada; Department of Bioengineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Michael I
authors.initials: MI
authors.lastname: Webb
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Walsby
authors.affiliation: Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON P3E 2C6, Canada. Electronic address: ssiemann@laurentian.ca.
authors.forename: Stefan
authors.initials: S
authors.lastname: Siemann
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry / Q000633:toxicity*; D002460:Cell Line; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008264:Macrophages / Q000187:drug effects; D008670:Metals / Q000737:chemistry*; D051379:Mice
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008670:Metals; C030325:anthrax toxin; D003300:Copper
keywords: Anthrax toxin; Apoprotein; Copper; Electron paramagnetic resonance (EPR); Metal substitution; Metalloprotease