Abstract
The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. 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, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended 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 binding 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 screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.
Highlights
The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells
Our results show that whereas the mode of activation for edema factor is distinctly different from that seen in mammalian adenylyl cyclases, these two classes of enzymes are likely to share a common catalytic mechanism
Our model is derived from the following observed constraints. (i) The 60-kDa monomeric EF3 fragment of edema factor consists of a highly structured core domain and of a loosely structured, approximately 160-residue long, C-terminal domain. (ii) The C-terminal region acquires a well defined structure upon CaM binding to EF3. (iii) The C-terminal region is essential for full CaM activation of edema factor. (iv) CaM has an extended conformation while bound to EF3. (v) Whereas both domains of CaM are required for the activation of edema factor, calcium binding in the C-terminal domain plays a privileged role in activation of edema factor when compared with calcium binding in the N-terminal domain
Summary
G-protein, guanine nucleotide-binding regulatory protein; CaM, calmodulin; EF, edema factor; EF3, C-terminal 60-kDa edema factor; EF3-N, N-terminal 43-kDa of EF3 (aa 291– 640); EF3-C, C-terminal 19-kDa EF3 (aa 640 – 800); MLCK, myosin light chain kinase; M13, synthetic peptide corresponding to the CaMbinding region of smooth muscle MLCK; Gs␣, the ␣-subunit of G-protein that stimulates adenylyl cyclase; AC, adenylyl cyclase; PAGE, polyacrylamide gel electrophoresis; 1,5-IAEDANS, N-iodoacetyl-NЈ-(5-sulfo1-naphthyl)ethylenediamine (AEDANS when attached to a protein); DAB, 4-dimethyl-aminophenylazophenyl-4Ј-maleimide; aa, amino acids; Mes, 4-morpholineethanesulfonic acid. The N-terminal portion of this 60-kDa region is similar to the adenylyl cyclase exotoxins from B. pertussis and P. aeruginosa, with 34 and 29% sequence identity, respectively. In contrast to mammalian adenylyl cyclases that contain a catalytic core composed of two homologous domains, edema factor and its related exotoxins have no internal sequence repeat and are active as monomers [12]. Binding of calcium ions has been shown to induce the movement of a pair of helices in a two EF-hand domain This leads to an opening of the domain and an exposure of a hydrophobic pocket, which becomes a target interaction site. The long central helix (residues 65–93) of CaM, which connects the two domains in the crystal structure of calcium-bound CaM, is disrupted This enables the C- and N-terminal domains to clamp the helical peptide, bringing the two domains of CaM close to each other. Our results show that whereas the mode of activation for edema factor is distinctly different from that seen in mammalian adenylyl cyclases, these two classes of enzymes are likely to share a common catalytic mechanism
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