Evidence That Histidine Protonation of Receptor-Bound Anthrax Protective Antigen Is a Trigger for Pore Formation

Abstract

The protective antigen (PA) component of the anthrax toxin forms pores within the low pH environment of host endosomes, through mechanisms that are poorly understood. It has been proposed that pore formation is dependent on histidine protonation. In previous work, we biosynthetically incorporated 2-fluorohistidine (2-FHis), an isosteric analog of histidine with a significantly reduced pKa (~1), into PA, and showed that the pH-dependent conversion from the soluble prepore to a pore was unchanged. However, we also observed that 2-FHisPA was nonfunctional in the ability to mediate cytotoxicity of CHO-K1 cells by LF N -DTA, and was defective in translocation through planar lipid bilayers. Here, we show that the defect in cytotoxicity is due to both a defect in translocation and, when bound to the host cellular receptor, an inability to undergo low pH-induced pore formation. Combining X-ray crystallography with hydrogendeuterium (H-D) exchange mass spectrometry, our studies lead to a model in which hydrogen bonds to the histidine ring are strengthened by receptor binding. The combination of both fluorination and receptor binding is sufficient to block low pH-induced pore formation.Anthrax toxin is an AB toxin secreted by Bacillus anthracis and is required for disease pathogenesis (1). The B component, the protective antigen (PA), is an 83 kDa, four domain protein that binds to the extracellular von Willebrand factor A (vWA) integrin-like I domain 1 To whom correspondence should be addressed: Tel: (316) Fax: (316) 978-3431; Jim.Bann@wichita.edu. 2 Current address: Department of Biological Sciences, University of Texas at El Paso, El Paso, Texas 79968-0519, USA 3 Current address: Department of Chemistry, University of Missouri-Columbia, 125 Chemistry Building, 601 S. College Avenue, Columbia, MO 65211-7600. Supporting informationSupplementary figures of translocation of LFN-DTA, a simulated-annealing omit map for the β1-β2 and β3-β4 loops, a figure showing the crystal contact between R344 and E224 and mass spectra of peptides used in the H-D exchange experiments. This material is free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 August 24. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript of either one of two identified host cell receptors, anthrax toxin receptor 1/tumor endothelial marker 8 (ANTXR1/TEM8) or anthrax toxin receptor 2/capillary morphogenesis protein 2 (ANTXR2/CMG2) (2,3). Binding of PA to the receptor vWA domain is followed by cleavage of PA into two fragments of 63 kDa and 20 kDa, by a cell-surface furin protease (4), leading to the spontaneous formation of a ring-shaped heptameric (PA 63 ) 7 structure termed the prepore (5). Formation of the prepore creates binding sites for the enzymatic A components, edema factor (EF) and lethal factor (LF), which bind with a stoichiometry of three EF or LF's per prepore (6). In addition, PA has been shown recently to form functional octa...

Domain Flexibility Modulates the Heterogeneous Assembly Mechanism of Anthrax Toxin Protective Antigen

“…the numbering convention of 1ACC is retained.) While our structure and the three reported monomeric structures, PA (PDB 1ACC), 23 the ANTXR2-bound PA (PDB 1T6B), 17 and the 2-fluoro-histidine substituted PA (PDB 3MHZ), 54 align with close agreement with a root mean square (RMS) < 1, A number of surface-loop regions not modeled in 1ACC are present in the PA ΔMIL structure. Residues 99-102 and 512-515 are not modeled in 1ACC; however, they are present in 1T6B, while the latter is present in 3MHZ.…”

A Receptor-based Switch that Regulates Anthrax Toxin Pore Formation

“…Furthermore, it is not clear if the receptor remains attached following pore conversion and, if so, how it remains attached. Evidence supporting dissociation has come from co-immunoprecipitation experiments [23] and from previous NMR studies [24], [25]. On the other hand, evidence in favor of receptor attachment has come from other co-immunoprecipitation studies [19], [26], from NMR binding studies performed with a fragment (Domain 4) of PA [27], and from the finding that the presence of a receptor seems to influence voltage-dependent inactivation and small molecule inhibition properties of the newly formed pore [28].…”

Anthrax toxin protective antigen integrates poly-γ- d -glutamate and pH signals to sense the optimal environment for channel formation

“…Recent studies suggest PA-ANTXR2 interactions begin to dissociate at pH 6 (18). Histidine protonation within PA (15) and at H121 in the ANTXR2 domain (4) may trigger channel formation, but 2-fluorohistidine (19,20) and mutagenesis (21) studies suggest histidine protonation may be important for dissociation from ANTXR2 but not for channel formation in solution. An electron microscopy (EM) structure of the channel supports the hypothesis (3, 4) that D2 and D4 separate as the channel forms (8).…”

“…3C), 3TEX, 3TEY, and 3TEZ (30), and 3Q8B and 3MHZ (20). We compared 4EE2 and 3TEW, as the latter is the highest-resolution structure of the native D2-D4 interface.…”