Edema Factor

Edema factor (EF) is encoded by the cya locus on the pXO1 plasmid [10]. The gene has been cloned [31,32] and sequenced [33].  It encodes a 2400-bp open reading frame, of which 99 bp encode a 33-amino-acid signal peptide and 2301 bp encode an A/T-rich (71%), cysteine-free, 767-amino-acid (88.8-kDa) mature secreted protein. 

EF was the first component of anthrax toxin shown to have an enzymatic activity.  Leppla [34] noted that cholera toxin, which activates eukaryotic adenylate cyclase by ADP ribosylation, can cause the formation of skin edema similar to that formed by edema toxin when injected subcutaneously into rabbits [35].  The effects of cholera toxin are readily assayed in CHOK1A cells, which undergo characteristic morphological changes shortly after toxin treatment [36].  Leppla [34] observed that EF plus PA induced a similar morphological response.  This could be competitively inhibited by the addition of LF [34], reflecting the fact that EF and LF bind the same site on PA.  Direct measurement of cAMP in the cells indicated that the addition of EF plus PA could cause as much as a 200-fold increase over nonstimulated levels and that this increase could be prevented by the simultaneous addition of an excess of LF.  However, unlike cholera toxin, EF appeared not to ADP-ribosylate adenylate cyclase, but was itself found to possess adenylate cyclase activity [34].  In vitro, purified EF has no activity, but the addition of nanomolar levels of calmodulin can activate EF in a Ca²⁺-dependent fashion [37].

The tertiary structures of the catalytic portion of EF alone and in complex with calmodulin have been solved to resolutions of 2.6 Å and 2.95 Å, respectively [38].  The catalytic portion of EF is composed of three domains: C_A (amino acids 294–309) and C_B (amino acids 350–489), which together form the active site, as well as a helical domain (amino acids 660-800) which is connected to C_A by a linker.  Calmodulin inserts at a position between C_A and the helical domain.  As a consequence of calmodulin binding, the helical domain rotates about and encircles calmodulin, locking it in position.  The binding of calmodulin and rotation of the helical domain alters the ordering of three switches—two from regions outside the globular domains that form the interface between the helical domain and C_A in unbound EF, and a third contained within C_A—so that they can participate in forming a substrate binding pocket to bind and position ATP.