A truncated Bordetella pertussis cya gene product wds expressed in Escherichia coli and purified by affinity chromatography on calmodulin-agarose. Trypsin cleavage of the 432-residue recombinant protein ( M , = 46 659) generated two fragments of 28 kDa and 19 kDa. These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin-binding properties. The 28-kDa peptide, corresponding to the N-terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein. The 19-kDa peptide, corresponding to the C-terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl-calmodulin. T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure. The two fragments reassociated into a highly active calmodulin-dependent species. Reassociation was enhanced by calmodulin itself, which 'trapped' the two complementary peptides into a stable, native-like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.Bordetella pertussis adenylate cyclase is synthesized as a large bifunctional precursor of 1706 amino acid residues [l]. The N-terminal segment of the protein (400 residues) has calmodulin-activated ATP-cyclizing activity, while the rest of the molecule is thought to be responsible for the haemolytic activity of the pathogen [l, 21. The large precursor, purified from extracts of B. pertussis as a 200-kDa protein [3 -51, is found in culture media most often in low-molecular-mass forms of 50, 45 or 43 kDa [6-81. Tryptic cleavage of these low-molecular-mass forms of adenylate cyclase yields two complementary fragments of 25 kDa and 18 kDa [9]. By combining genetic and biochemical information it has been shown that the 25-kDa fragment harbors the active site, whereas the 18-kDa fragment corresponds mostly to the calmodulinbinding domain of the enzyme [lo, 111.
Calmodulin‐activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13–24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site‐directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin‐binding properties of mutated proteins showed no important differences when compared to the wild‐type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3′‐anthraniloyl‐2′‐deoxyadenosine 5′‐triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP‐utilizing enzymes, are essential for binding the Mg(2+)‐nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide‐binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.
The sequence situated around Trp242 in Bordetella pertussis adenylate cyclase, a bifunctional protein of 1706 amino acid residues, forms the core of the calmodulin-binding site. Peptides varying in size and in affinity for calmodulin, and preserving the same sequence around Trp242 were analyzed by timeresolved fluorescence spectroscopy. Their dynamic properties were compared to those of the catalytic domain of B. pertussis adenylate cyclase corresponding to the first 400 amino acid residues of the protein and in which the Trp69 residue was replaced by Phe. The heterogeneity of the fluorescence intensity decays of Trp242 is likely due to the existence of conformers in equilibrium as is suggested by the effect of trifluoroethanol both on the secondary structure content and the lifetime distributions. Binding to calmodulin leads to striking effects on the lifetime distribution profiles by selecting a major excited state population and therefore one major conformer. Trp242 still presents some degree of rotational freedom in the complexes. The reduction of rotational freedom is more important for the shorter peptides than for the longest one. A similar selection of one major conformer with the same lifetime was also observed for the Trp242 in the mutant protein when bound to calmodulin, as in the complexes with the peptides. We conclude that the site of interaction of B. pertussis adenylate cyclase with calmodulin has similar conformational flexibility as that evidenced in the isolated peptides. This property of the molecule allows a better adjustment of the enzyme upon interaction with calmodulin.
A truncated, 432 residue long, Bordetella pertussis adenylate cyclase expressed in Escherichia coli was analyzed for intrinsic fluorescence properties. The two tryptophans (Trp69 and Trp242) of adenylate cyclase, each situated in close proximity to residues important for catalysis or binding of calmodulin (CaM), produced overlapping fluorescence emission bands upon excitation at 295 nm. CaM, alone or in association with low concentrations of urea, induced important modifications in the spectra of adenylate cyclase such as shifts of the maxima and change in the shape of the bands. From these changes and from the fluorescence spectrum of a modified form of adenylate cyclase, in which a valine residue was substituted for Trp242, it was deduced that, upon binding of CaM to the wild-type adenylate cyclase, only the environment of Trp242 was affected. The fluorescence maximum of this residue, which is more exposed to the solvent than Trp69 in the absence of CaM, is shifted by 13 nm to shorter wavelength upon interaction of protein with its activator. Trypsin cleaved adenylate cyclase into two fragments, one carrying the catalytic domain, and the second carrying the CaM-binding domain (Ladant et al., 1989). The isolated peptides conserved most of the environment around their single tryptophan residues, as in the intact adenylate cyclase, which suggests that the two domains of truncated B. pertussis adenylate cyclase also conserved most of their three-dimensional structure in the isolated forms.
A truncated, 541-residue-long, Bacillus anthracis adenylate cyclase was expressed in Escherichia coli. The purified protein (CYA 62) exhibited catalytic and CaM-binding properties identical with those of the wild-type enzyme secreted by B. anthracis. The analysis of the secondary structure of the CYA 62 protein by Fourier transform infrared spectroscopy and circular dichroism revealed the dominance of beta-type structure. The protein shows a relatively low thermal stability with the midpoint denaturation temperature at 45 degrees C. A catalytically inactive variant of CYA 62 in which Gln substituted for Lys-346 (CYA 62 K346Q) was comparatively analyzed for its secondary structure and thermal stability, as well as ligand-binding properties with fluorescent derivatives of ATP and calmodulin. The K346Q variant of CYA 62 has a similar secondary structure and comparable calmodulin binding properties to those of the parent protein and exhibits only slightly reduced thermal stability (the apparent midpoint denaturation temperature is at 43 degrees C). Despite these similarities, the binding of 3'-anthraniloyl-2'-deoxy-ATP (a fluorescent ATP analogue) to the modified protein is severely impaired, from which we conclude that the prime function of Lys-346 in the wild-type enzyme from B. anthracis is to ensure tight binding of the nucleotide substrate to the active site.
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