Hélène Munier

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

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.

Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase

This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain. Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM). The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure. Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure. The peptide is flexible in solution, being in equilibrium between random coil and helical structures. Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state. The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation. No elements of tertiary structure organization were detected by the present experiments. The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin.

New fluorescent and photoactivable analogs acting on nucleotide binding enzymes

Abstract We describe a seven-step synthesis of 8-azido-3′-O-anthraniloyl-2′dADP and 2′dATP from 8-azido-2′deoxyadenosine. These compounds can be used as fluorescent and photoactivable probes for the nucleotide-binding site of kinases or cyclases.

Structural and Functional Organization of the Catalytic Domain of a Bacterial Toxin: bordetella Pertussis Adenylate Cyclase

Bacterial toxins frequently appear as multidomain proteins in which each domain corresponds to a well-defined function, such as recognition of cell surface receptors, translocation across membrane(s) and interaction with specific target(s).