Assessing the Contribution of Cavity Density to Protein Partial Molar Expansivity by High-Pressure NMR

Abstract

peats, characteristic of the Repeats in ToXin (RTX) proteins, and an N-terminal proteolytic (passenger) domain. The exoprotease is secreted through a type I secretion system (TISS) composed of a tripartite complex formed by an ABC-transporter, a membrane fusion protein, and a TolC-like outer membrane protein. TISS substrates vary widely in size and function and include toxins, lipases, and proteases. The conserved feature of the substrate proteins is the presence of one or more RTX motifs. Previously, we have demonstrated that calcium regulates multiple conformations of AP. Calcium-induced RTX domain folding serves to chaperone the folding of the protease domain. Here we show that disruption of the calcium-binding sites alters both the affinity and cooperativity of calcium-induced folding, measured in the RTX domain and in the full-length protease, and that the binding sites are not isoenergetic. We have also evaluated the role of calcium in the secretion of the protease, as previous studies have suggested that calcium may facilitate protease secretion. Protein secretion was efficient when the passenger domain was maintained in an unfolded conformation and secreted into medium with high calcium concentrations. Secretion efficiencies decreased with mutations in the RTX domain and with passenger domains that were stable and folded in the bacterial cytoplasm. From these results we conclude calcium regulates protease conformation and may contribute to secretion efficiencies by maintaining specific protein conformations during translocation. These results provide a basis for understanding the calcium-associated secretion RTX proteins from multiple bacterial pathogens.