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.
Highlights
Disorder-to-order transition plays a key role in the biological function of many proteins that contain intrinsically disordered regions
We review below a series of recent works that have contributed to provide a better understanding of the structure-function relationships of the RTX proteins [1,2,3,4]
We showed that the RTX proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium: they adopt pre-molten globule conformations and exhibit a strong time-averaged apparent hydration, due in part to the internal electrostatic repulsions between negatively charged residues, as revealed by the high mean net charge of the apo-polypeptide [4]
Summary
Disorder-to-order transition plays a key role in the biological function of many proteins that contain intrinsically disordered regions. Iwaki and co-workers previously showed that the last 217 C-terminal residues (encompassing the block V and its N and C-terminal flanking regions) constitute an autonomous domain required for both cytotoxic and hemolytic activities of the adenylate cyclase toxin [28]. They showed that truncated CyaA proteins lacking the last 75 C-terminal residues did not exhibit any toxic activity. A polypeptide encompassing the RTX motifs of block V together with the N- and C-terminal flanking sequences was able to bind up to seven calcium ions per polypeptide with an affinity in the millimolar range This polypeptide was able to acquire secondary structure in the presence of calcium [21]. 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
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