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Purification and Biophysical Characterization of the Core Protease Domain of Anthrax Lethal Factor

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Purification and Biophysical Characterization of the Core Protease Domain of Anthrax Lethal Factor Biochemical and Biophysical Research Communications 396 (2010) 643–647 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Purification and biophysical characterization of the core protease domain of anthrax lethal factor Petros V. Gkazonis a, Georgios A. Dalkas a, Christos T. Chasapis a, Alexios Vlamis-Gardikas b, Detlef Bentrop c, Georgios A. Spyroulias a,* a Department of Pharmacy, University of Patras, GR-26504 Patras, Greece b Department of Biochemistry, Foundation for Biomedical Research (BRFAA), Academy of Athens, GR-11527 Athens, Greece c Institute of Physiology II, University of Freiburg, D-79108 Freiburg, Germany article info abstract Article history: Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a Received 9 April 2010 90 kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn2+ binding Available online 8 May 2010 site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given Keywords: the importance of the interaction between LF and substrate for the development of anti-anthrax agents as Anthrax lethal factor well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties LF catalytic site of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant Zn metalloprotease expression and purification of a C-terminal part of LF (LF ) that harbours the enzyme’s core protease Recombinant protein expression 672–776 1 13 15 NMR spectroscopy domain. The biophysical characterization and backbone assignments ( H, C, N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn2+, suitable for high resolution structural analysis by NMR. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction family (MEKs/MKKs) close to their N-termini, thus inactivating them, preventing MKK signalling [5,6] and ultimately causing Inhalation anthrax is the most fatal form of the disease anthrax apoptosis [7]. Recently the interaction of LeTx and in particular caused by the uptake of Bacillus anthracis spores by pulmonary LF with its MKK substrates has attracted considerable scientific macrophages. The spores are carried through lymph nodes, germi- interest due to the potential use of B. anthracis in bioterrorism. In nating en route and spread throughout the host’s bloodstream, addition, recent reports implicate the exclusive specificity of LF where they live and proliferate as extracellular pathogens, causing for MKKs in the regulation of MKK-dependent tumour cell signal- massive septicemia and toxemia resulting in systemic effects that ling and proliferation, stimulating thus the study of native and can cause the host’s death [1]. engineered forms of the enzyme for tumour targeting [8]. For this The virulence factor of B. anthracis is a secreted toxin (anthrax purpose, the stability of the polypeptide chain in denaturing condi- toxin, ATx) [2–4] that consists of three proteins, none of which is tions and the unfolding/refolding properties of the apo and metal- toxic on its own: the 83 kDa protective antigen (PA), the 89 kDa lated enzyme catalytic site are of immense interest. calmodulin-activated edema factor adenylate cyclase (EF) and the The X-ray structure of LF revealed a mainly a-helical protein with 90 kDa anthrax lethal factor (LF). EF combined with PA (PA–EF) four domains [9]. The C-terminal domain IV (residues 552–776) form the edema toxin, while LF combined with PA form the Lethal comprises the catalytic center of the enzyme and contains the active Toxin (LeTx). All three components act in synergy to kill host mac- site in a 40 Å long groove created by the vestigial NAD-binding pock- rophages. Once endocytosed, LF acts as a Zn2+ dependent endopep- et of domain II and the interface between domains II, III, and IV. The tidase cleaving with remarkable specificity members of the MAPKK groove has an overall negative charge, as it contains clusters of glu- tamic and aspartic acid residues [9]. Two Zn2+-binding motifs (H686E687F688G689H690 and E735F736F737A738E739) that can together Abbreviations: LeTx, anthrax lethal toxin; LF, anthrax lethal factor; CD, circular bind to Zn2+ with a 1:1 stoichiometry are located in the catalytic dichroism; VKK, MAPK (mitogen-activated protein kinase) kinases; NMR, nuclear pocket of LF. The motifs are separated by a spacer of 44 residues magnetic resonance; HSQC, heteronuclear single quantum coherence. [10]. The active site Zn2+ is coordinated tetrahedrally by a water * Corresponding author. Fax: +30 2610969950. E-mail address: [email protected] (G.A. Spyroulias). molecule and the three side chains of His686, His690, and Glu735. With 2+ URL: http://www.bionmr.upatras.gr (G.A. Spyroulias). respect to its Zn -binding site, LF is classified in the MA clan and 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.144 644 P.V. Gkazonis et al. / Biochemical and Biophysical Research Communications 396 (2010) 643–647 M34 family of Zn-metalloproteases [11]. To comprehend the acetohydroxamic acid (AHA). After concentration to 500 ll, the structure–activity relationship of the catalytic site of LF and sample was loaded onto a Superose 12 10/300GL size exclusion map its atomic level dynamics one needs to monitor the catalytic column using an ÄKTA FPLC™ system (Amersham PharmaciaÒ). 2+ site behaviour towards Zn binding, substrate recognition and LF672–776 was eluted with a high salt buffer (Tris–HCl 20 mM, EDTA interaction in vitro. 5 mM, NaCl 500 mM, pH 7.8) to prevent ionic interactions with Here we report the heterologous expression, purification and residual GST and thrombin and finally dialyzed against application preliminary biophysical characterization of a 105 a.a. C-terminal buffer. construct of the LF catalytic domain (LF672–776 hereafter), harbour- 2+ ing the enzyme’s Zn -binding (active) site (core protease domain). 2.3. Purification and biophysical characterization The recombinant LF catalytic domain was expressed as a GST fusion protein in Escherichia coli and purified from cell extract as Purity of the apo protein samples was checked by Tris–glycine a soluble protein in its apo form to prevent autoproteolysis and SDS–PAGE and automated electrophoresis (Experion, Bio-RadÒ). 2+ to study the Zn binding effects on the structure and dynamics The concentration of the protein samples was estimated by Bradford of the molecule. NMR and CD data showed that LF672–776 is a folded assay using BSA protein standards and confirmed by Lab on Chip protein with secondary and tertiary structure. Heteronuclear NMR automated electrophoresis (Experion). All further characterization allowed an almost complete sequence specific assignment of was carried out in the presence of 5 mM EDTA to retain LF672–776 13C/15N nuclei. 2+ in apo form. Binding of Zn to apoLF672–776 was achieved by dialysis against Tris–HCl 20 mM, NaCl 0.1 M, AHA 0.2 mM, ZnCl2 1 mM, pH 7.2, and metal ion excess was removed by dialysis against metal-free 2. Materials and methods buffer. Protein samples for NMR experiments were prepared in 90% H 0 (50 mM KPi, 5 mM EDTA, 0.2 M AHA, pH 7.2) and 10% D Oat 2.1. Plasmids, bacterial strains, and media 2 2 0.6–0.8 mM protein concentration. Samples of labelled protein were checked on a Bruker Avance DRX 400 MHz NMR Spectrometers A portion of LF full length gene (nucleotides encoding 105 C-ter- while 2D/3D homo- and heteronuclear NMR spectra were recorded minal amino acids) was amplified using primers F 107 BamH1 (TAT at 298 K on a Bruker Avance 600 MHz NMR Spectrometer, equipped ATT AGG ATC CAA AGG TGT AGA ATT AAG G) and LF EcoRC 6His with a cryogenically cooled pulsed field gradient triple resonance (ATT GAA TTC TTA GTG ATG ATG GTG ATG ATG TGA GTT AAT probe (TXI). Sequence specific HN,N,Ca and C0 assignments were AAT GAA CTT AAT CTG ATCG), using as template full length LF in obtained from the following experiments: 2D [1H–15N]-HSQC, 3D pGEX-2TK [12]. The amplified fragment was inserted in vector HNCA, 3D HN(CO)CA, 3D CBCA(CO)NH, 3D CBCANH and 3D HNCO. pGEMT and sequenced. The desired clones were inserted as a Internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as BamH1-EcoR1 fragment into expression vector pGEX-4T1 (Amer- a chemical shift reference for 1H. All NMR data were processed with sham BiosciencesÒ), in frame with the existing GST gene of the vec- the Bruker XWINNMR or TOPSPIN software and analyzed with the tor. In this manner, the portion of the LF gene is downstream the programs XEASY [13] and CARA [14]. The backbone 1H, 13C and one coding for GST, the two peptides joined by a thrombin cleavage 15N chemical shifts have been deposited in the BioMagResBank site. Top10 chemically competent E. coli strains (InvitrogenÒ) were (http:/www.bmrb.wisc.edu) under the accession number 16735. used as host strains for plasmid propagation and subcloning appli- Secondary structure prediction based on the chemical shifts of back- cations, while BL21(DE3)Gold (StratageneÒ) strains were used as bone 1H and 13C nuclei was performed using CSI (Chemical Shift expression hosts. All bacterial strains were routinely cultured in Index; http://www.bionmr.ualberta.ca/bds/software/csi/latest/csi. Luria Bertani medium (LB) in liquid or solid medium
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