Oedema factor, a calmodulin-activated adenylyl cyclase, is important in the pathogenesis of anthrax. Here we report the X-ray structures of oedema factor with and without bound calmodulin. Oedema factor shares no significant structural homology with mammalian adenylyl cyclases or other proteins. In the active site, 3'-deoxy-ATP and a single metal ion are well positioned for catalysis with histidine 351 as the catalytic base. This mechanism differs from the mechanism of two-metal-ion catalysis proposed for mammalian adenylyl cyclases. Four discrete regions of oedema factor form a surface that recognizes an extended conformation of calmodulin, which is very different from the collapsed conformation observed in other structures of calmodulin bound to effector peptides. On calmodulin binding, an oedema factor helical domain of relative molecular mass 15,000 undergoes a 15 A translation and a 30 degrees rotation away from the oedema factor catalytic core, which stabilizes a disordered loop and leads to enzyme activation. These allosteric changes provide the first molecular details of how calmodulin modulates one of its targets.

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C 1 and C 2 ). The first cytoplasmic domain of type I enzyme (IC 1 ) and the second cytoplasmic domain of type II enzyme (IIC 2 -⌬3, a construct in which 36 N-terminal amino acids of the C 2 region are deleted) were expressed and purified to homogeneity. Alone, each had no adenylyl cyclase activity; however, mixing of the two domains in vitro resulted in G s␣ -and forskolin-activated enzyme activity. The turnover number for G s␣ -and forskolin-stimulated enzyme activity of the complex between IC 1 and IIC 2 -⌬3 was 8.2 s ؊1 . The concentration of IIC 2 -⌬3 to achieve half-maximal activation of IC 1 was 0.8 and 1.3 M when stimulated by forskolin and G s␣ , respectively. The concentration of IIC 2 -⌬3 needed to complex with IC 1 was reduced 10-fold (0.08 M) when the enzyme was activated by both forskolin and G s␣ , suggesting that G s␣ and forskolin increased the affinity of the two cytoplasmic domains for each other.The enzymatic activity of adenylyl cyclase is the key step in regulating the intracellular cAMP concentration upon stimulation of a variety of hormones, neurotransmitters, and other regulatory molecules. There are at least nine distinct mammalian adenylyl cyclases which have a similar structure (Fig. 1A) (1-11). This includes two intensely hydrophobic domains (M 1 and M 2 ) and two ϳ40-kDa cytoplasmic domains (C 1 and C 2 ). The C 1 and C 2 domains contain sequences (C 1a and C 2a ) that are similar to each other and to other adenylyl and guanylyl cyclases (12, 13). Each isoform of adenylyl cyclase has its own distinct tissue distribution and unique regulatory properties, providing modes for different cells to respond diversely to similar stimuli (12,14).Membrane-bound adenylyl cyclases are expressed in small quantities, and the enzyme is labile and difficult to manipulate in detergent-containing solutions. To facilitate biochemical and structural analysis, a soluble adenylyl cyclase has been constructed by linking the C 1a and C 2a domains of type I and type II adenylyl cyclases, respectively (15). The resulting protein is sensitive to activation by G s␣ 1 and forskolin and to inhibition by P-site inhibitors, indicating the essential roles of C 1a and C 2a domains for catalysis and regulation. In this paper, we describe the expression and purification of the C 1a and C 2a domains of type I and type II adenylyl cyclase, respectively. Alone, each has no adenylyl cyclase activity; however, mixing of the two domains in vitro results in G s␣ -and forskolin-activated enzyme activity. EXPERIMENTAL PROCEDURESPlasmids-For construction of the expression plasmid vector pProEx-HAH6, the NcoI and EcoRI 4.9-kb fragment of pProEx-1 (Life Technologies, Inc.) was ligated with the phosphorylated linkers (5Ј-CATGCATCACCATCACCATCACGCGGCCGCCTACCCGTATGATGT-CCCGGATTACGCCGGAATTCCCATGGC and 5Ј-AATTGCCATGGGA-ATTCCGGCGTAATCCGGGACATCATACGGGTAGGCGGCCGCGTG-TGGTGATGGTGATG). Proper insertion of cDNA at the NcoI site of pProEx-HAH6 vector would result in the expres...

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 Nnor 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.cAMP is a key second messenger that modulates a particularly diverse set of physiological responses including sugar and lipid metabolism, cell differentiation, apoptosis, neuronal activity, and ion homeostasis. Certain pathogenic bacteria have evolved exotoxins that severely alter the internal cAMP levels of infected cells. These exotoxins work through two common mechanisms. The first is to ADP-ribosylate the ␣-subunits of heterotrimeric G-proteins.

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C 1 and C 2 ), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C 2 domain is catalytically active and the C 1 domain is not; the role of the C 1 domain is to promote the catalytic activity of the C 2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C 2 domains, but not among the C 1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30 -100-fold reduction in K cat of adenylyl cyclase. However, the same mutations do not affect the K m for ATP, the half-maximal concentration (EC 50 ) for the C 2 domain of type II adenylyl cyclase to associate with the C 1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC 50 for forskolin to maximally activate enzyme activity with or without G s␣ . This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C 1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in K cat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.The activity of mammalian adenylyl cyclase, the enzyme that converts ATP to cAMP, is the key step in modulating intracellular cAMP concentration in response to extracellular stimulation by hormones, neurotransmitters, and odorants. Nine isoforms of mammalian adenylyl cyclases have been cloned to date, and they belong to a rapid expanding cyclase family that includes class III adenylyl cyclases 1 and guanylyl cyclases (1).All nine isoforms have a similar structure, including two intensely hydrophobic domains (M 1 and M 2 ) and two 40-kDa cytoplasmic domains (C 1 and C 2 ). The two cytoplasmic domains contain sequences (C 1a and C 2a ) that are homologous to each other and to class III adenylyl cyclases and guanylyl cyclases. Each isoform of adenylyl cyclase not only has distinct patterns of tissue expression but also has unique responses to extracellular and intracellular stimuli (1-3). In common, each adenylyl cyclase can be activated by the G protein 2 ␣ subunit, designated as G s␣ , and each can be inhibited by certain adenosine analogues (P-site inhibitors). However, there are several subtype-specific regulators, including forskolin, G protein ␤␥ subunits, the ␣ subunit of G i , G o , and G z , Ca 2ϩ ion, Ca 2ϩ -calmodulin, Ca 2ϩ -calcineurin, cAMP-dependent protein kinase, and PKC (for review, see Refs. 1-3). Certain ones of these regulators (e.g. ␤␥) can be either stimulatory or inhibitory...