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Evidence that histidine protonation of receptor-bound anthrax protective antigen is a trigger for pore formation
Wimalasena, D. Shyamali Janowiak, Blythe E. Lovell, Scott Miyagi, Masaru Sun, Jianjun Zhou, Haiying Hajduch, Jan Pooput, Chaya Kirk, Kenneth L. Battaile, Kevin P.
Wimalasena, D. Shyamali
Janowiak, Blythe E.
Lovell, Scott
Miyagi, Masaru
Sun, Jianjun
Zhou, Haiying
Hajduch, Jan
Pooput, Chaya
Kirk, Kenneth L.
Battaile, Kevin P.
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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 non-functional in the ability to mediate cytotoxicity of CHO-K1 cells by LFN-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.
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2010-08-24
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ACS
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Wimalasena_ACS_2010.pdf
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Wimalasena, D. S., Janowiak, B. E., Lovell, S., Miyagi, M., Sun, J., Zhou, H., … Bann, J. G. (2010). Evidence that histidine protonation of receptor-bound anthrax protective antigen is a trigger for pore formation. Biochemistry, 49(33), 6973–6983. http://doi.org/10.1021/bi100647z