Abstract
SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.