An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis

The edema factor exotoxin produced by Bacillus anthracis fs an adenylyl cyc lase that is activated by calmodulin (CaM) at resting state calcium concent rations in infected cells. A C-terminal 60-kDa fragment corresponding to th e catalytic domain of edema factor (EF3) was cloned, overexpressed in Esche richia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetell a pertussis and Pseudomonas aeruginosa, is highly resistant to protease dig estion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, an d both fragments were required to reconstitute full CaM-sensitive enzyme ac tivity. Fluorescence resonance energy transfer experiments using a double-l abeled CaM molecule were performed and indicated that CaM adopts an extende d conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM -dependent protein kinase type II. Mutations in each of the four calcium bi nding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic scr een probing loss-of-function mutations of EF3 and site-directed mutations b ased on the:homology of the edema factor family revealed a conserved pair o f aspartate residues and an arginine that: are important for catalysis. Sim ilar residues are essential for di-metal-mediated catalysis in mammalian ad enylyl cyclases and a family of DNA polymerases and nucleotidyltransferases . This suggests that edema factor may utilize a similar catalytic mechanism .