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

Darragh P O’Brien,Ana-Cristina Sotomayor-Pérez,Sébastien Brier,Alexandre Chenal,Véronique Hourdel,Daniel Ladant,Mahmoud Ghomi,Julia Chamot-Rooke,Dominique Durand,Belen Hernandez,Patrice Vachette

doi:10.1038/srep14223

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.

Highlights

Disorder-to-order transitions play a key role in the biological functions of many proteins that contain intrinsically disordered regions[1,2]
It is immediately apparent from the Rg reduction, P(r) and Kratky profiles that Apo-RTX domain (RD) is essentially unstructured and undergoes a major transition to a much more compact conformation upon calcium binding
We propose structural models (Fig. 6 and Supplementary Movie), based on combined Small Angle X-ray Scattering (SAXS), Raman spectroscopy and Hydrogen/Deuterium eXchange-Mass Spectrometry (HDX-MS) data, for both the unstructured, calcium-free Apo-state and the folded calcium-bound Holo-state of the RTX domain (RD) of the B. pertussis CyaA toxin. This data, in line with prior biophysical and thermodynamic studies, strongly supports the view that the disordered state of the RTX motifs within the bacteria may facilitate protein secretion through the type 1 secretion system (T1SS) while in the extracellular environment, calcium triggers folding of the toxin into its cytotoxic active form

Summary

Introduction

Disorder-to-order transitions play a key role in the biological functions of many proteins that contain intrinsically disordered regions[1,2]. The conclusion of this model-independent analysis of the two scattering curves is that Apo-RD behaves as an unstructured polymeric chain with no structured globular regions, but with local, transient structural elements, while in the presence of calcium, Holo-RD adopts a compact, folded and anisometric conformation.

Results

Conclusion