Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy

Abstract

The interaction of the adenylate cyclase catalytic domain (AC) of the Bordetella pertussis major exotoxin with its activator calmodulin (CaM) was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located in different regions of AC: tryptophan residues (W69 and W242), a nucleotide analogue (3¢-anthraniloyl-2¢-deoxyadenosine 5¢-triphosphate, Ant-dATP) and a cysteinespecific probe (acrylodan). CaM binding elicited large changes in the dynamics of W242, which dominates the fluorescence emission of both AC and AC-CaM, similar to that observed for isolated CaM-binding sequences of different lengths [Bouhss, A., Vincent, M., Munier, H., Gilles, A.M., Takahashi, M., Baˆrzu, O., Danchin, A. & Gallay, J. (1996) Eur. J. Biochem. 237, 619-628]. In contrast, AntdATP remains completely immobile and inaccessible to the solvent in both the AC and AC-CaM nucleotide-binding sites. As AC contains no cysteine residue, a single-Cys mutant at position 75 was constructed which allowed labeling of the catalytic domain with acrylodan. Its environment is strongly apolar and rigid, and only slightly affected by CaM. The protein's hydrodynamic properties were also studied by fluorescence anisotropy decay measurements. The average Brownian rotational correlation times of AC differed significantly according to the probe used (19 ns for W242, 25 ns for Ant-dATP, and 35 ns for acrylodan), suggesting an elongated protein shape (axial ratio of % 1.9). These values increased greatly with the addition of CaM (39 ns for W242, 60-70 ns for Ant-dATP and 56 ns for acrylodan). This suggests that (a) the orientation of the probes is altered with respect to the protein axes and (b) the protein becomes more elongated with an axial ratio of % 2.4. For comparison, the hydrodynamic properties of the anthrax AC exotoxin were computed by a mathematical approach (HYDROPRO), which uses the 3D structure [Drum, C.L., Yan, S. A change in axial ratio is also observed on CaM binding, but in the reverse direction from that for AC: from 1.7 to 1.3. The mechanisms of activation of the two proteins by CaM may therefore be different.

Structural basis for the interaction of Bordetella pertussis adenylyl cyclase toxin with calmodulin

“…No electron density can be found between A225 and L233 of CyaA-ACD, which is the trypsin-sensitive site that divides CyaA-ACD into the N-terminal T25 (25 kDa tryptic fragment) and the Cterminal T18 (Ladant, 1988). Consistent with the fluorescence anisotropy decay data, the C-CaM-bound CyaA-ACD has an elongated shape ( Figure 1) (Gallay et al, 2004). C-CaM adopts the calcium-loaded, open conformation.…”

Adenylate Cyclase Toxin (ACT) fromBordetella hinzii: Characterization and Differences from ACT ofBordetella pertussis

“…It is believed that calmodulin is able to activate ACT by binding to the toxin with a high affinity (0.2 nM), causing a conformational change in a flexible loop between the ATPand CaM-binding subdomains and bringing together the amino acids necessary for ACT to bind and catalyze ATP into cAMP (23,26). In an attempt to corroborate our Blue-Sepharose competition results, we compared the calmodulin activation between ACT from both Bordetella species.…”

Temperature-accelerated molecular dynamics gives insights into globular conformations sampled in the free state of the AC catalytic domain

“…Extensive biophysical analyses on CyaA activation, however, provided the following information 6 : (i) the CaM bound and unbound AC conformations show a very similar secondary structure content, (ii) the unbound conformation displays a more globular shape than the bound state, with hydrodynamic parameters similar to that of a sphere, (iii) the transition to a more globular shape might be caused by a docking of the α helix H against the CA domain, with an angular displacement of the helix axis that could reach 90°. Conversely, a study of the fluorescence of Trp‐242, located in the helix H, has revealed in the isolated AC a large internal mobility of this residue, which seems to indicate that the isolated AC undergoes a conformational equilibrium between several protein conformations.…”