Shui-Zhong Yan

Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin

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 15u2009Å translation and a 30° 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.

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 byBacillus 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 inEscherichia 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 andPseudomonas 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.

Three discrete regions of mammalian adenylyl cyclase form a site for Gsalpha activation.

The interaction between the α subunit of G protein Gs (Gsα) and the two cytoplasmic domains of adenylyl cyclase (C1 and C2) is a key step in the stimulation of cAMP synthesis by hormones. Mutational analysis reveals that three discrete regions in the primary sequence of adenylyl cyclase affect the EC50values for Gsα activation and thus are the affinity determinants of Gsα. Based on the three-dimensional structure of C2·forskolin dimer, these three regions (C2 α2, C2 α3/β4, and C1β1) are close together and form a negatively charged and hydrophobic groove the width of an α helix that can accommodate the positively charged adenylyl cyclase binding region of Gsα. Two mutations in the C2 α3/β4 region decrease theV max values of Gsα activation without an increase in the EC50 values. Since these three regions are distal to the catalytic site, the likely mechanism for Gsα activation is to modulate the structure of the active site by controlling the orientation of the C2 α2 and α3/β4 structures.

The Regulation of Type 7 Adenylyl Cyclase by Its C1b Region and Escherichia coli Peptidylprolyl Isomerase, SlyD*

Mammalian membrane-bound adenylyl cyclase consists of two highly conserved cytoplasmic domains (C1a and C2a) separated by a less conserved connecting region, C1b, and one of two transmembrane domains, M2. The C1a and C2a domains form a catalytic core that can be stimulated by forskolin and the stimulatory G protein subunit α (Gαs). In this study, we analyzed the regulation of type 7 adenylyl cyclase (AC7) by C1b. The C1a, C1b, and C2a domains of AC7 were purified separately. Escherichia coli SlyD protein, a cis-transpeptidylprolyl isomerase (PPIase), copurifies with AC7 C1b (7C1b). SlyD protein can inhibit the Gαs- and/or forskolin-activated activity of both soluble and membrane-bound AC7. Mutant forms of SlyD with reduced PPIase activity are less potent in the inhibition of AC7 activity. Interestingly, different isoforms of mammalian membrane-bound adenylyl cyclase can be either inhibited or stimulated by SlyD protein, raising the possibility that mammalian PPIase may regulate enzymatic activity of mammalian adenylyl cyclase. Purified 7C1b-SlyD complex has a greater inhibitory effect on AC7 activity than SlyD alone. This inhibition by 7C1b is abolished in a 7C1b mutant in which a conserved glutamic acid (amino acid residue 582) is changed to alanine. Inhibition of adenylyl cyclase activity by 7C1b is further confirmed by using 7C1b purified from an E. coli slyD-deficient strain. This inhibitory activity of AC7 is also observed with the 28-mer peptides derived from a region of C1b conserved in AC7 and AC2 but is not observed with a peptide derived from the corresponding region of AC6. This inhibitory activity exhibited by the C1b domain may result from the interaction of 7C1b with 7C1a and 7C2a and may serve to hold AC7 in the basal nonstimulated state.

Construction of soluble adenylyl cyclase from human membrane-bound type 7 adenylyl cyclase.

Adenylyl cyclase is the sole enzyme to synthesize cyclic AMP (cAMP), a key, second messenger that regulates diverse physiological responses including sugar and lipid metabolism, olfaction, and cell growth and differentiation. Most adenylyl cyclase activity in mammalian tissues is found in plasma membrane preparations with the exception of the reproductive organs, which have both membrane-associated and soluble forms of adenylyl cyclase. To date, nine membrane-bound and one soluble adenylyl cyclases from mammals have been cloned and characterized. Each isoform has its own pattern of tissue distribution and regulation. This chapter describes how to make a soluble adenylyl cyclase from the human type 7 isoform.