Evidence that histidine protonation of receptor-bound anthrax protective antigen is a trigger for pore formation.
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.
Wimalasena
D Shyamali
DS
Department of Chemistry, Wichita State University, Wichita, Kansas 67260, USA.
Janowiak
Blythe E
BE
Lovell
Scott
S
Miyagi
Masaru
M
Sun
Jianjun
J
Zhou
Haiying
H
Hajduch
Jan
J
Pooput
Chaya
C
Kirk
Kenneth L
KL
Battaile
Kevin P
KP
Bann
James G
JG
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*
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D011522:Protons; D011956:Receptors, Cell Surface; C030325:anthrax toxin; D006639:Histidine