title: 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 analogue of histidine with a significantly reduced pK(a) ( approximately 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 hydrogen-deuterium (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.
authors.affiliation: Department of Chemistry, Wichita State University, Wichita, Kansas 67260, USA.
authors.forename: D Shyamali
authors.initials: DS
authors.lastname: Wimalasena
authors.forename: Blythe E
authors.initials: BE
authors.lastname: Janowiak
authors.forename: Scott
authors.initials: S
authors.lastname: Lovell
authors.forename: Masaru
authors.initials: M
authors.lastname: Miyagi
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sun
authors.forename: Haiying
authors.initials: H
authors.lastname: Zhou
authors.forename: Jan
authors.initials: J
authors.lastname: Hajduch
authors.forename: Chaya
authors.initials: C
authors.lastname: Pooput
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Kirk
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Battaile
authors.forename: James G
authors.initials: JG
authors.lastname: Bann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000378:metabolism*; D001408:Bacillus anthracis / Q000378:metabolism* / Q000472:pathogenicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021381:Protein Transport; D011522:Protons; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011522:Protons; D011956:Receptors, Cell Surface; C030325:anthrax toxin; D006639:Histidine