High metal substitution tolerance of anthrax lethal factor and characterization of its active copper-substituted analogue Suet Y. Lo a , Crystal E. Säbel a , Michael I. Webb b,c , Charles J. Walsby b , Stefan Siemann a, ⁎ a Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON P3E 2C6, Canada b Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada c Department of Bioengineering, University of California, Berkeley, CA 94720, USA abstract article info Article history: Received 19 March 2014 Received in revised form 27 May 2014 Accepted 16 June 2014 Available online 24 June 2014 Keywords: Anthrax toxin Apoprotein Copper Electron paramagnetic resonance (EPR) Metalloprotease Metal substitution 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 experi- ments 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. © 2014 Elsevier Inc. All rights reserved. 1. Introduction Anthrax is an infectious bacterial disease caused by Bacillus anthracis, and has been the topic of considerable interest as an animal and human pathogen, as well as a potential component of biological weapons [1]. Three proteins, protective antigen (PA), edema factor (EF), and lethal factor (LF), collectively known as the anthrax toxin, play a pivotal role in the manifestation of the disease [2]. PA (in its heptameric or octameric form) is a pore-forming protein which mediates the entry of LF and EF from the endosome into the host cell cytosol [3–7]. EF is a calcium and calmodulin-dependent adenylyl cyclase responsible for the generation of supraphysiological amounts of cyclic AMP, thus resulting in a disruption of water homeostasis [8,9]. LF is a zinc- dependent metalloendopeptidase responsible for the cleavage of most members of the mitogen-activated protein kinase kinase (MAPKK) family of signalling proteins near their N-termini [10–13]. Furthermore, LF has recently been shown to facilitate inflammasome activation and macrophage death by removing an N-terminal segment from NOD- like receptor protein 1 (Nlrp1) [14–17]. The zinc ion in LF is coordinated to the side chains of His686, His690, and Glu735, and to a water molecule which serves as the nucleophile in the peptide bond hydrolysis reaction [13,18]. In addition, Glu687, which is part of the thermolysin-like HExxH consensus motif [19], has been proposed to serve as a general base in the catalytic mechanism of LF by activating and orienting the zinc-bound water molecule for proper nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond [13]. In view of the aforementioned amino acid residues involved in zinc coordination and water activation, LF can be classified as a gluzincin [19]. Initial investigations on the metal requirement of LF using reconsti- tution assays involving apoLF have revealed the protein's stringent re- quirement for Zn 2+ for catalytic function. Indeed, apoLF preparations supplemented with other transition metal ions such as Mn 2+ , Co 2+ , Ni 2+ and Cu 2+ were found not to regain a significant degree of catalytic competence (typically below 2% of the activity of native ZnLF) [20], although some of these ions were found to effectively compete with Zn 2+ for protein binding in earlier radiolabeling studies [21]. Further- more, Ca 2+ and Mg 2+ have previously been shown to be required to restore (some of) the activity of apoLF by Zn 2+ and other divalent tran- sition metal ions [20,22]. However, recent studies have revealed the re- quirement for these alkaline earth metals not to be an inherent feature Journal of Inorganic Biochemistry 140 (2014) 12–22 ⁎ Corresponding author. Tel.: +1 705 675 1151; fax: +1 705 675 4844. E-mail address: ssiemann@laurentian.ca (S. Siemann). http://dx.doi.org/10.1016/j.jinorgbio.2014.06.009 0162-0134/© 2014 Elsevier Inc. All rights reserved. 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