Effect of 2-Fluorohistidine Labeling of the Anthrax Protective Antigen on Stability, Pore Formation, and Translocation ² D. Shyamali Wimalasena, ‡ John C. Cramer, § Blythe E. Janowiak, | Stephen J. Juris, |,# Roman A. Melnyk, |,£ D. Eric Anderson, ⊥ Kenneth L. Kirk, § R. John Collier, | and James G. Bann* ,‡ Department of Chemistry, Wichita State UniVersity, Wichita, Kansas 67260-0051, Laboratory of Bioorganic Chemistry, and Proteomics and Mass Spectrometry Facility, National Institute of Diabetes and DigestiVe and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, and Department of Microbiology and Molecular Genetics, HarVard Medical School, Boston, Massachusetts 02115 ReceiVed August 29, 2007; ReVised Manuscript ReceiVed October 10, 2007 ABSTRACT: The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pK a (∼1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LF N -DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA 83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA 63 ) 7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin- activated PA was able to permeabilize liposomes to K + and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo. Vegetative Bacillus anthracis secretes a toxin that is comprised of a tripartite set of proteins that includes the protective antigen (PA), 1 edema factor (EF), and lethal factor (LF). The transport of EF and LF into the cell, a process that is critical for the pathogenesis of anthrax, can only occur through a pore formed by the PA (1). PA is a four-domain, 83 kDa protein (Figure 1A) that recognizes on host cells the von Willebrand factor A domains (VWA) of two integrin- like receptors, anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) (2-4). Binding of PA to the host cell receptor leads to the proteolytic cleavage of PA by a furin-like protease on the cell surface, releasing the first 167 amino acid residues of domain 1. This processing event is followed by oligomerization of the remaining cell-bound 63 kDa segment into a donut-shaped heptameric structure called the prepore (5). The formation of the prepore creates binding sites for EF and LF, which bind with a stoichiometry of 3 per heptameric prepore (6, 7). The toxin is then internalized into an early endosome, which is trafficked to a late ² This work supported by NIH U54 AI057160 to the Midwest Regional Center of Excellence for Biodefense and Emerging and Infectious Disease Research (MRCE), an NIH IDEA/COBRE-PSF award through the University of Kansas (J.G.B.), and by NIH Grant AI22021 (R.J.C.). Research at NIH (K.L.K., J.C.C., and D.E.A.) was supported by the intramural research funds of NIDDK. * To whom correspondence should be addressed. Phone: 316-978- 7373. Fax: 316-978-3431. E-mail: Jim.Bann@wichita.edu. ‡ Wichita State University. § Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases. ⊥ Proteomics and Mass Spectrometry Facility, National Institute of Diabetes and Digestive and Kidney Diseases. | Harvard Medical School. # Current address: Departments of Biology and Chemistry, Central Michigan University, Mount Pleasant, MI, 48859. £ Current address: Merck Research Laboratories, Merck Frosst Canada & Co., Inc., Department of Biochemistry, Office 10-3-220, 16711 TransCanada Highway, Kirkland QC, Canada H9H 3L1. 1 Abbreviations: Ala, alanine; ANTXR2, anthrax toxin receptor 2 (formerly CMG2, capillary morphogenesis protein 2); ANTXR1, anthrax toxin receptor 1 (formerly ATR-TEM8, anthrax toxin receptor- tumor endothelial marker 8); Asn, asparagine; Asp, aspartate; BisTris, bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane; CD, circular dichroism; DPhPC, 1,2-diphytanoyl-sn-glycerol-3-phosphocholine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; EF, edema factor; 2-FHis, 2-fluorohistidine; Glu, glutamate; HEPES, N-(2-hydroxyethyl)pipera- zine-N′-(2-ethane sulfonic acid); IPTG, isopropyl--D-thiogalactopy- ranoside; K + , potassium ions; LF, lethal factor; LFN, residues 1-263 of lethal factor; LFN-DTA, fusion of LFN and the catalytic domain of diphtheria toxin; Lys, lysine; MIDAS, metal ion-dependent adhesion site; N, native folded state; I, intermediate folded state; U, unfolded state; PA, protective antigen; PA 83, protective antigen precursor; PA63, a single monomer of the heptamer of protective antigen; (PA63)7, the heptameric form of the protective antigen; PCR, polymerase chain reaction; Phe, phenylalanine; VWA, the von Willebrand factor A domain of ANTXR2; WT, wild-type. 14928 Biochemistry 2007, 46, 14928-14936 10.1021/bi701763z CCC: $37.00 © 2007 American Chemical Society Published on Web 11/29/2007