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Article

Effect of 2-Fluorohistidine Labeling of the Anthrax Protective Antigen on Stability, Pore Formation, and Translocation^†

Supporting Info

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

Biochemistry, 2007, 46 (51), pp 14928–14936

DOI: 10.1021/bi701763z

Publication Date (Web): November 29, 2007

†

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.

‡

Wichita State University.

Laboratory of Bioorganic Chemistry, 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.

Proteomics and Mass Spectrometry Facility, National Institute of Diabetes and Digestive and Kidney Diseases.

To whom correspondence should be addressed. Phone: 316-978-7373. Fax: 316-978-3431. E-mail: Jim.Bann@wichita.edu.

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 PA₈₃ protein in the pH 5−6 range, the pH dependence of prepore-to-pore conversion of (PA₆₃)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.