A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen.
Bacillus anthracis secretes three proteins, which associate in binary combinations to form toxic complexes at the surface of mammalian cells. Receptor-bound protective antigen (PA) is proteolytically activated, yielding a 63 kDa fragment (PA(63)). PA(63) oligomerizes into heptamers, which bind edema factor (EF) or lethal factor (LF) to form the toxic complexes. We undertook a quantitative analysis of the interactions of EF with PA(63) by means of surface plasmon resonance (SPR) measurements. Heptameric PA(63) was covalently bound by amine coupling to an SPR chip, or noncovalently bound via a C-terminal hexahistidine tag on the protein to Ni(2+)nitrilotriacetate groups on the chip. Values of k(on) and k(off) for EF at 23 degrees C were approximately 3 x 10(5) M(-)(1) s(-)(1) and (3-5) x 10(-)(4) s(-)(1), respectively, giving a calculated K(d) of (1-2) x 10(-)(9) M. A similar value of K(d) (7 x 10(-)(10) M) was obtained when we measured the binding of radiolabeled EF to receptor-bound PA(63) on the surface of L6 cells (at 4 degrees C). Each of these analyses was also performed with LF and LF(N) (the N-terminal 255 residues of LF), and values obtained were comparable to those for EF. The similarity in the dissociation constants determined by SPR and by measurements on the cell surface suggests that the presence of the receptor does not play a large role in the interaction between PA(63) and EF/LF.
Elliott
J L
JL
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mogridge
J
J
Collier
R J
RJ
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000276:immunology; D001408:Bacillus anthracis / Q000737:chemistry* / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry*; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D018000:Receptors, Peptide / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013696:Temperature
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D018000:Receptors, Peptide; C030325:anthrax toxin; C070309:anthrax toxin receptors