Anthrax Protective Antigen:  Efficiency of Translocation Is Independent of the Number of Ligands Bound to the Prepore

Abstract

Heptameric anthrax protective antigen (termed prepore), which assembles at the mammalian cell surface, competitively binds edema factor (EF) and/or lethal factor (LF). It then transports them to an acidic intracellular compartment and mediates their translocation across the membrane to the cytosol. Steric constraints limit to three the number of molecules of EF and/or LF that can bind simultaneously to prepore. To determine whether the number of ligand molecules bound per heptamer affects the efficiency of translocation, we measured the low-pH-triggered translocation of the radiolabeled protective antigen (PA(63))-binding domain of LF ((35)S-LF(N)) across the plasma membrane of CHO-K1 cells as a function of the degree of saturation of the prepore. The fraction translocated remained constant at approximately 0.4 as (35)S-LF(N) was varied from nil through saturating concentrations. The same constant value was observed when we held (35)S-LF(N) at a saturating concentration and varied the number of functional ligand sites per prepore by changing the ratio of wild-type PA to a ligand-binding mutant. Thus, prepore containing only a single ligand-binding site is capable of translocating its cargo as efficiently as one containing multiple binding sites. The results as a whole imply that heptamers with one, two, or three ligands bound translocate their ligands with the same efficiency, indicating that each ligand molecule is translocated independently from the others.

Anthrax Edema Factor, Voltage-dependent Binding to the Protective Antigen Ion Channel and Comparison to LF Binding

“…For most experiments, the His 6 tag was removed by enterokinase treatment. His 6 -EF exhibited an even higher affinity for binding to the PA 63 channel similar as has previously been found for a truncated form of LF, LF N , or LF 263 (11,23,33). The half-saturation constant for His 6 -EF binding to the PA 63 channel was on average 0.16 nM (K ϭ 6.25⅐10 9 1/M) at an ionic strength of 150 mM KCl; which means that the stability constant for binding for His 6 -EF was roughly a factor of ten higher than that without the His 6 tag (see Table 1).…”

“…Binding of His 6 -EF to the PA 63 channels was much less ionic strength-dependent than EF. The influence of the partially positively charged His 6 tag on EF binding indicates again that the interaction between the N-terminal end of EF and the PA 63 channels is based on ion-ion interactions similarly as discussed in the case of LF N (23).…”

“…This result indicated that channels, which were not blocked before by EF at zero voltage closed as a result of the higher voltage. This result suggested an increase of the stability constant of binding up to very high voltages an effect that has not been observed with full-length LF and the truncated form LF N (11,23).…”

“…The molecular masses of LF and EF are very similar, which means that it is impossible that three EF molecules bind to the PA 63 channel with the same affinity to the binding site. The transport efficiency of the toxins into the target cells is independent from the number of bound toxin molecules to the PA heptamer indicating the independent translocation of them (23).…”

“…The negatively charged PA 63 channel interacts with positively charged quaternary ammonium ions, which have a structure similar to that of the N-terminal ends of LF and EF (20 -22). Studies with a truncated form of LF, LF N with a length of 263 amino acids suggest that LF interacts with its N-terminal end with the PA 63 channel (23). LF N is also able to block PA 63 channels in a voltage-dependent and reversible way, which suggested that the PA 63 channel could be the pathway of delivery of LF into the cytosol of the target cells (24).…”