Calcium-Induced Folding and Stabilization of the Intrinsically Disordered RTX Domain of the CyaA Toxin

Abstract

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. Its C-terminal region, the receptor-binding domain (RD), contains ∼40 calcium-binding Repeat in ToXin (RTX) motifs, which are characteristic of many virulence factors of pathogenic bacteria. We previously showed that RD is intrinsically disordered in the absence of calcium and acquires its functional three-dimensional structure upon calcium binding. To gain further insight into the physicochemical properties of RD, we characterized its calcium-induced conformational and stability changes by combining spectroscopic approaches. We show that RD, in the absence of calcium, adopts premolten globule conformations, due in part to the strong internal electrostatic repulsions between the negative charges of the aspartate-rich polypeptide sequence. Accordingly, sodium is able to screen these electrostatic repulsions, allowing a partial compaction of the polypeptide, whereas calcium triggers a strong compaction as well as the acquisition of secondary and tertiary structures in a highly cooperative manner. The differential sensitivity of the calcium-loaded state to guanidinium- and urea-induced denaturations provides further evidence that electrostatic interactions play a critical role in the folding and stability of RD. These results provide new insights into the folding/function relationship of the RTX motifs.

Allosteric Activation of Bordetella pertussis Adenylyl Cyclase by Calmodulin

“…The thermodynamic stability of the AC proteins was investigated by following their urea-induced denaturation (at 25°C, in buffer A), which was monitored by tryptophan fluorescence spectroscopy as described previously (30), using an FP-6200 spectrofluorimeter (Jasco, Japan) in a Peltier-thermostated cell holder, with a 1-cm path length quartz cell (101.QS, Hellma). The thermodynamic parameters (urea concentration required to unfold half the population of native proteins ([urea]1 ⁄ 2 ), the cooperativity (m), and free energy (⌬G)) were deduced from the fluorescence data, as described previously (30). (15), we first checked the conservation of the atomic contacts established by these residues in the crystallographic structures of the AC⅐C-CaM complex (13).…”

Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

“…The 675-residue RD consists of ∼40-45 glycine-and aspartate-rich repeat in toxin (RTX) motifs that are involved in calcium binding (29). In the absence of calcium the CyaA RD does not fold into a well-defined 3D structure, but instead exists in a premolten globule ensemble of conformations (30). Like other intrinsically disordered polypeptides it contains a high content of random coils and is highly hydrated (30)(31)(32).…”

“…In the absence of calcium the CyaA RD does not fold into a well-defined 3D structure, but instead exists in a premolten globule ensemble of conformations (30). Like other intrinsically disordered polypeptides it contains a high content of random coils and is highly hydrated (30)(31)(32). Upon binding calcium, the CyaA RD folds into a stable β-roll structure that facilitates interaction with a cell surface receptor and the translocation of an N-terminal adenylate cyclase domain across the plasma membrane (32)(33)(34)(35)(36).…”

Bacterial Translocation Ratchets: Shared Physical Principles with Different Molecular Implementations

“…[86] Accordingly, Ca 2þ ions are required for folding and hence, cytotoxicity of the T1SS secreted proteins. [87][88][89][90] Recent studies provide evidence for an intramolecular translocation ratchet powered by folding of the RTX-domain: Within the "channel-tunnel" system of the T1SS that spans the entire cell envelope, the secreted proteins are transported in an unfolded state, as expected from the channel dimensions [91] ( Figure 4B). The unfolded protein is transported in a C to N direction, leading to initial exposure of the C-terminus and thus the first RTX repeat segment.…”