Calcium-induced Folding of Intrinsically Disordered Repeat-in-Toxin (RTX) Motifs via Changes of Protein Charges and Oligomerization States

Daniel Ladant,Ana Cristina Sotomayor-Pérez,Alexandre Chenal

doi:10.1074/jbc.m110.210393

Daniel Ladant, Ana Cristina Sotomayor-Pérez + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m110.210393

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Abstract

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 so-called RTX (repeat-in-toxin) motifs found in many virulence factors secreted by numerous gram-negative pathogenic bacteria: RTX proteins are natively disordered in the absence of calcium but fold upon calcium binding. The adenylate cyclase toxin (CyaA) produced by Bordetella pertussis, the causative agent of whooping cough, contains ∼40 RTX motifs organized in five successive blocks separated by non-RTX flanking regions. This RTX domain mediates toxin binding to its eukaryotic cell receptor. We previously showed that the last block of the RTX domain, block V, which is critical for CyaA toxicity, exhibits the hallmarks of intrinsically disordered proteins in the absence of calcium. Moreover, the C-terminal flanking region of CyaA block V is required for its calcium-induced folding. Here, we describe a comprehensive analysis of the hydrodynamic and electrophoretic properties of several block V RTX polypeptides that differ in the presence and/or length of the flanking regions. Our results indicate that the length of the C-terminal flanking region not only controls the calcium-induced folding but also the calcium-induced multimerization of the RTX polypeptides. Moreover, we showed that calcium binding is accompanied by a strong reduction of the net charge of the RTX polypeptides. These data indicate that the disorder-to-order transition in RTX proteins is controlled by a calcium-induced change of the polypeptide charges and stabilized by multimerization.

Highlights

For proteins, commonly designated as natively or intrinsically disordered proteins, that are lacking ordered structures under physiological conditions [1,2,3,4,5,6,7,8]
The original name was first coined to group four virulence factors secreted by Escherichia coli, Bordetella pertussis, Pasteurella hemolytica, and Actinobacillus sp., that contain a series of tandemly repeated RTX sequences at their C-terminal end
They all encompass the core of the last block of RTX motifs from CyaA but differ in the presence of either the N-terminal or the C-terminal (C) flanking region, the latter being further subdivided into short CS and long CL regions

Summary

Introduction

For proteins, commonly designated as natively or intrinsically disordered proteins, that are lacking ordered structures under physiological conditions [1,2,3,4,5,6,7,8]. We have recently begun to explore the biophysical mechanisms at work in this process in a newly described class of intrinsically disordered protein that contains so-called RTX repeated motifs and in which the disorder-to-order transition is triggered by the binding of calcium ions (9 –11). These motifs are found in many virulence factors secreted by numerous Gram-negative pathogenic bacteria [12, 13]. The disordered conformations of RTX motifs in the low calcium environment of the bacterial cytosol may facilitate the protein secretion through the type I secretion system machinery, whereas in the external medium, calcium binding could trigger the folding of the proteins into their active conformations for host-pathogen interactions (9, 19 –21)

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