Lethal Factor

Lethal factor (LF) is encoded by the lef locus on the pXO1 plasmid [10]. The gene has been cloned [39] and sequenced [40], and it encodes a 2427-bp open reading frame, of which 99 bp encode a 33-amino-acid signal peptide and 2328 bp encode an A/T-rich (70%), cysteine-free, 776–amino-acid (90.2-kDa) mature secreted protein.  The linear sequence of LF may be divided into three regions: the N-terminal 254 amino acids, which show a high degree of similarity to the N-terminus of EF; residues 282–383, which are composed of five imperfect 19-amino-acid repeats; and the remainder of the protein, which shows no apparent homology to known proteins. 

Based on the high degree of homology between the N-termini of LF and EF, Bragg and Robertson [40] first predicted that this region likely contained elements required for PA binding.  This was later confirmed when Quinn et al. [41] showed that whereas mutation at the N-terminus eliminates binding to PA and toxicity, mutations at the C-terminus eliminate toxicity without affecting PA binding.  Moreover, it has been shown that fusion proteins in which amino acids 1–254 of LF (LF1–254; LFn) are fused to catalytic domains of other toxins can bind to PA and reach the cytosol of mammalian cells in active conformation [42,43,44,45,46].  Mutations in the region containing the imperfect repeats interfere with LF synthesis, suggesting that this region may play an essential role in generating tertiary structure [41]. 

The tertiary structure of LF has been solved to a resolution of 2.2 Å; the molecule is 100 Å tall and 70 Å wide at its base [47].  LF is composed of four domains.  Domain I comprises the previously described N-terminal portion, which binds PA.  Domain II (residues 263-297 and 385-550) shows structural similarity with the ADP-ribosylating toxin of Bacillus cereus but lacks the residues required for NAD binding and catalysis.  Domain III (residues 303-382) is inserted into domain II and contains a series of four tandem imperfect repeats of a helix-turn element present in domain II.  Acidic residues in domain III form specific contacts with the basic N-termini of MEKs.  Domain IV (residues 552-776) has limited structural homology to thermolysin and contains the catalytic core.  Interaction with the N-terminus of MEK is mediated by domains II, III, and IV, which together create a 40 Å long groove into which MEK fits.