Molecular Crowding Effects on the CyaA Toxin RTX Domain: Implication for Toxin Secretion

Abstract

Ligand‐induced disorder‐to‐order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered domains. Here, we present data on an RTX (« Repeat in ToXin ») protein, RCL, an intrinsically disordered protein (IDP) that folds upon calcium binding. RTX motifs are calcium‐binding nonapeptide sequences that are found in more than 250 virulence factors secreted by Gram‐negative pathogenic bacteria. The RCL protein is a RTX region of the CyaA toxin, one of the major virulence factors secreted by Bordetella pertussis, the causative agent of the whooping cough. Using a combination of biophysical approaches, we showed that RCL exhibits the hallmarks of IDP in the absence of calcium. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of RCL. We further showed that the crowding agent Ficoll70 did not affect the structural content of the apo‐state and holo‐state of RCL but increased protein affinity for calcium and stabilizes both states of RCL, increasing their half‐melting temperature (ΔTm), without affecting enthalpy changes. The power law dependence of the ΔTm increase on the volume fraction allowed the estimation of the Flory exponents of the thermally unfolded states. Altogether, our data suggest that, in the apo‐state as found in the crowded bacterial cytosol, RTX proteins adopt extended unfolded conformations that may facilitate protein export by the secretion machinery. Subsequently, both calcium gradient across bacterial cell wall and molecular crowding enhance the calcium‐dependent folding and stability of RTX proteins once secreted in the extracellular milieu.