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Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA(63), forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA(63)-channels. Here, in experiments with artificial lipid bilayer membranes, EF(N) and LF(N) show block of PA(63)-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF(N), increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA(63)-channel.

The lethal factor (LF) of Bacillus anthracis is a Zn2+-endopeptidase specific for the MAPK-kinase family of proteins. The catalytic zinc atom is coordinated by a first shell of residues including the two histidines and the glutamate of the zinc-binding motif HExxH and by Glu-735. A characteristic feature of LF is the presence, within the second shell of residues, of a tyrosine (Tyr-728) in close proximity (3.3 A) to the zinc atom. To investigate the role of Tyr-728 and Glu-735, LF mutants with one or both of these two residues replaced by Ala were cloned, expressed, and purified from Escherichia coli. A fourth mutant was obtained by replacing Tyr-728 with Phe. Spectroscopic analysis of these mutants indicates that they fold in the same way as the parental molecule and that zinc stabilizes the structure of LF. These mutants have neither proteolytic activity nor in vivo toxicity. The possible role of Tyr-728 in catalysis is discussed.