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

Abstract

Many Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound Holo-state. Apo-RD behaves as a disordered polymer chain comprising several statistical elements that exhibit local rigidity with residual secondary structure. Holo-RD is a folded multi-domain protein with an anisometric shape. RTX motifs thus appear remarkably adapted to the structural and mechanistic constraints of the secretion process. In the low calcium environment of the bacterial cytosol, Apo-RD is an elongated disordered coil appropriately sized for transport through the narrow secretion machinery. The progressive folding of Holo-RD in the extracellular calcium-rich environment as it emerges form the T1SS may then favor its unidirectional export through the secretory channel. This process is relevant for hundreds of bacterial species producing virulent RTX proteins.

Post‐translational acylation controls the folding and functions of the CyaA RTX toxin

“…These data have direct implications for CyaA secretion. We previously suggested that in the low calcium environment of the bacterial cytosol, RD adopts disordered conformations favorable for transport through the type 1 secretion machinery and folds upon binding calcium in the extracellular, calcium-rich environment (4,32,34,39,41,42). Our present data now extend this model to the full-length CyaA toxin.…”

“…Finally, the C-terminal 701 residues (1006-1706) correspond to the cell receptor-binding domain, RTX domain (RD), which harbors ;40 copies of a glycine-and aspartate-rich nona-peptide repeat, characteristic of the RTX bacterial cytolysins. In the absence of calcium, these RTX motifs are intrinsically disordered but undergo a disorder-to-order transition upon calcium binding (4,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42). The RTX motifs themselves constitute the primary sites of calcium binding within the protein (3,31,43,44).…”

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

“…MEMHDX was evaluated using our recently published differential HDX-MS dataset generated with the C-terminal R epeat-in-toxin D omain (RD, 701 residues) of the CyaA toxin (O'Brien et al , 2015). Briefly, we used HDX to identify and locate secondary structural elements in the intrinsically disordered Apo-RD protein, and followed its transition to a more compact and folded state upon calcium binding.…”

“…peptide 395–401). Results generated with MEMHDX were compared with those manually obtained for RD (O'Brien et al , 2015). Excellent agreement was observed between the automated software and the manual approach.…”

“…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). …”