Molecular Crowding Stabilizes Both the Intrinsically Disordered Calcium-Free State and the Folded Calcium-Bound State of an RTX Protein: Implication for Toxin Secretion

Abstract

Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered domains. Here, we present data on an RTX ( Repeat in ToXin ) protein, RCL, an IDP that folds upon calcium binding. RTX motifs are calcium-binding nonapeptide sequences that are found in more than 250 virulence factors secreted by Gram-negative pathogenic bacteria. Using a combination of biophysical approaches, we showed that RCL exhibits the hallmarks of intrinsically disordered proteins in the absence of calcium. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of RCL. Moreover, RCL is an attractive model to investigate the effect of molecular crowding because it offers the opportunity to characterize the crowding effects on the same protein under two drastically distinct folding states. Macromolecular crowding affects most chemical equilibria in living cells by sterically restricting the available space. We showed that the crowding agent Ficoll70 did not affect the structural content of the apo-state and holo-state of RCL but increased the protein affinity for calcium. Besides, Ficoll70 strongly stabilizes both states of RCL, increasing their half-melting temperature (ΔTm), without affecting enthalpy changes. The power law dependence of the ΔTm increase on the volume fraction allowed the estimation of the Flory exponent of the thermally unfolded states. Altogether, our data suggest that, in the apo-state as found in the crowded bacterial cytosol, RTX proteins adopt extended unfolded conformations that may facilitate protein export by the secretion machinery. Subsequently, calcium gradient across bacterial cell wall and crowding also enhances the calcium-dependent folding and stability of RTX proteins once secreted in the extracellular milieu.