RTX Calcium Binding Motifs Are Intrinsically Disordered in the Absence of Calcium

Abstract

The Repeat in Toxin (RTX) motif is a tandemly repeated calcium-binding nonapeptide sequence present in proteins that are secreted by the type I secretion system (T1SS) of Gram-negative bacteria. Here, we have characterized the structural and hydrodynamic properties of the RTX Repeat Domain (RD) of the CyaA toxin from Bordetella pertussis. This 701-amino acid long domain contains about 40 RTX motifs. We showed that, in the absence of calcium, RD was natively disordered, weakly stable, and highly hydrated. Calcium binding induced compaction and dehydration of RD, along with the formation of stable secondary and tertiary structures. The calcium-induced conformational switch between unfolded conformations of apo-RD and stable structures of holo-RD is likely to be a key property for the biological function of the CyaA toxin: in the low calcium environment of the bacterial cytosol, the intrinsically disordered character of the protein may facilitate its secretion through the secretion machinery. In the extracellular medium, calcium binding can then trigger the folding of the polypeptide into its functional state. The intrinsic disorder of RTX-containing proteins in the absence of calcium may thus be directly involved in the efficient secretion of proteins through T1SS.

Calcium, Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

“…Circular Dichroism Spectroscopy-CD spectra were recorded on an Aviv circular dichroism spectrometer model 215, equipped with a water-cooled Peltier unit as described elsewhere (17). CD measurements were carried out at a scan rate of 0.5 nm/s (step, 0.5 nm; integration time, 1 s) with a time constant of 100 ms and a bandwidth of 1 nm.…”

“…These motifs constitute the main Ca 2ϩ binding sites of the protein (16). The RTX motifs are intrinsically disordered in the absence of calcium (17)(18)(19)(20). The intrinsic disorder predictors (21)(22)(23)(24) (Fig.…”

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin

“…This is consistent with the findings of Pimenta et al (2005) who showed that the amount of HlyA secreted and the activity of HlyA increased in line with the extracellular Ca 2+ concentration over a millimolar range. Chenal et al (2009) indeed proposed that Ca 2+ -dependent folding of the RTX region of emerging HlyA proteins could contribute a nucleus for folding of the more proximal regions of the protein. Intriguingly, the geometry of the HlyA molecule -with conceivably at least two distinct types of folding pathway triggers, positioned towards the C terminus of the protein -the RTX Ca 2+ repeats and the Cterminal end itself, suggest that the C terminus may lead the way out of the translocator.…”

“…Interestingly, Chenal et al (2009) and Sotomayor Perez et al (2010) recently showed that milimolar concentrations of Ca 2+ ions in vitro promote the compaction of the normally disordered RTX region of a type I protein, together with increased formation of stable secondary and tertiary structures. This is consistent with the findings of Pimenta et al (2005) who showed that the amount of HlyA secreted and the activity of HlyA increased in line with the extracellular Ca 2+ concentration over a millimolar range.…”

“…It is also assumed that intracellular refolding of RTX proteins is prevented due to the low cytosolic Ca 2+ concentration (300 nM), despite extracellular Ca 2+ concentrations of up to 10 mM (Jones et al, 2002). Sanchez-Magraner et al (2007) demonstrated that in the presence of Ca 2+ , the b-roll structure of the RTX domain of HlyA is compacted and stabilized, and Chenal et al (2009) recently described similar effects of Ca 2+ on the otherwise disordered RTX domain of the adenylate cyclase toxin. These results and a further study (Herlax & Bakas, 2007) appear to point towards HlyA being a conformationally flexible molecule, whose properties are commensurate with secretion in a relatively unfolded state on the one hand and the requirement for radical conformational changes for insertion into membranes on the other.…”