Effect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocation.
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 pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-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.
Department of Chemistry, Wichita State University, Wichita, Kansas 67260-0051, USA.
D Shyamali
DS
Wimalasena
John C
JC
Cramer
Blythe E
BE
Janowiak
Stephen J
SJ
Juris
Roman A
RA
Melnyk
D Eric
DE
Anderson
Kenneth L
KL
Kirk
R John
RJ
Collier
James G
JG
Bann
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000031:analogs & derivatives* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D021381:Protein Transport
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; C514961:fluorohistidine; D006639:Histidine