A High‐Affinity Calmodulin‐Binding Site in the CyaA Toxin Translocation Domain is Essential for Invasion of Eukaryotic Cells

Abstract

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.

The role of four cholesterol-recognition motifs localized between amino acid residues 400-550 in regulating translocation and lytic activity of Adenylate Cyclase Toxin

“…By contrast, the molecular mechanisms and the structural elements involved in AC domain translocation are less clear, and remain a matter of intense research. Several models have been postulated the last years [27, 28]. One model posits that the ACT pore-forming activity is not implicated in the delivery of the AC domain across target cell membrane, and that on its way into the cytosol the translocating AC domain bypasses the cation-selective and lytic pore formed by ACT into the membrane [19, 27].…”

“…Furthermore, structural integrity of all the transmembrane helices of the HD were shown to be essential for AC domain translocation across the plasma membrane of both CD11b+ and CD11b-cells [19, 23, 24]. A second model posits that upon ACT insertion into the target cell membrane, a helical peptide extending from residue 454 to 484 interacts with the plasma membrane and destabilizes the lipid bilayer which would favour direct AC translocation across the lipid bilayer [28]. High affinity binding of that segment with calmodulin in the cell cytosol would then assist the irreversible translocation of the entire AC domain [28].…”

“…A second model posits that upon ACT insertion into the target cell membrane, a helical peptide extending from residue 454 to 484 interacts with the plasma membrane and destabilizes the lipid bilayer which would favour direct AC translocation across the lipid bilayer [28]. High affinity binding of that segment with calmodulin in the cell cytosol would then assist the irreversible translocation of the entire AC domain [28]. Data by others demonstrating the translocation of unrelated polypeptides fused to the ACT haemolysin moiety [29], weaken however reliability of this hypothesis.…”

Dynamics and structural changes of calmodulin upon interaction with its potent antagonist calmidazolium

“…Even a target enzyme, such as B. pertussis CyaA that is activated by interacting with the CaM C-terminal lobe only, 54,55,49 can be efficiently inhibited by CDZ. 35 This suggests that CDZ, by stabilizing the closed CaM globular conformation, should be capable of blunting all major CaM associations with most enzyme targets. Interestingly, this mode of binding is similar to that of TFP except that this drug, in contrast to CDZ, mainly contacts residues in the C-lobe of CaM.…”

“…Overall, binding of a second CDZ is unlikely to occur in solution in standard physiological assays in vitro or in cells (with CDZ below 20 μM) and it is therefore reasonable to assume that in these tests, binding of a single CDZ can fully block CaM activity. This is important to consider when experimentally probing CaM signaling function with CDZ at a concentration able to match the total CaM concentration in cell (estimated to range from 3 to 10 μM, depending on cell types 35 ).…”