Disorder-to-Order Transition in the CyaA Toxin RTX Domain: Implications for Toxin Secretion

Abstract

The past decade has seen a fundamental reappraisal of the protein structure-to-function paradigm because it became evident that a significant fraction of polypeptides are lacking ordered structures under physiological conditions. Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. This trait is exhibited by RTX (Repeat in ToXin) motifs found in more than 250 virulence factors secreted by Gram-negative pathogenic bacteria. We have investigated several RTX-containing polypeptides of different lengths, all derived from the Bordetella pertussis adenylate cyclase toxin, CyaA. Using a combination of experimental approaches, we showed that the RTX proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium. This intrinsic disorder mainly results from internal electrostatic repulsions between negatively charged residues of the RTX motifs. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the RTX proteins. We propose that the intrinsically disordered character of the RTX proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. These results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing RTX motifs, could be finely regulated by the calcium gradient across bacterial cell wall.

Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin

“…Thus, when the RTX repeats follow the ss through the TISS channel, relatively abundant Ca 2+ in the extracellular environment binds to the aspartate residues triggering concomitant folding. The secreted, folded, and now stabilized RTX motif would prevent back import and facilitate continued export and folding of the holotoxin via a molecular ratchet mechanism ( 34 – 38 ). In fact, Ca 2+ -dependent secretion has been suggested for other RTX toxins, including the Bordetella pertussis adenylate cyclase toxin and Escherichia coli pore-forming hemolysin ( 34 – 38 ).…”

MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets

“…A user-friendly interface developed with Shiny by RStudio facilitates the use of the application, allowing the user to easily visualize and compare the relative deuterium incorporation across the entire protein sequence and 3D structure. As a test system, we used the receptor-binding Repeat-in-ToXin (RTX) domain of CyaA produced by Bordetella pertussis , the causative agent of whooping cough, to pinpoint regions undergoing structural and conformational changes upon calcium binding (O'Brien et al , 2015; Sotomayor-Perez et al , 2015). …”

Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion

“…, Apo-form); they adopt disordered conformations mainly due to electrostatic repulsions between negatively charged residues 25 . Calcium binding triggers a strong reduction of the mean net charge, dehydration, compaction, folding and stabilization of secondary and tertiary structures of RTX proteins 26 .…”