Chaperone/usher machinery

Many virulence organelles of Gram-negative bacterial pathogens are assembled via the periplasmic chaperone/usher (CU) pathway. The assembly process is a complex task, involving secretion of organelle subunits via the two membranes and periplasm, subunit folding and assembly. In this thesis, the mechanism of the organelle subunit trafficking and assembly via the CU pathway was investigated at different steps, subunit capture by the chaperone (paper II), usher targeting (paper III), and transport through the usher (paper I), using the Caf1M/Caf1M CU system that assembles the Yersinia pestis F1 capsular antigen from Caf1 subunits. In paper II, we performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 subunit and analyzed the effect of the mutations on structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. We show that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enable Caf1M to rapidly capture/fold Caf1 subunit. The switch from the Caf1M-Caf1 contact to the Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M with improved chaperone function was engineered. In paper III, we elucidated the mechanism of the usher-targeting step at atomic resolution. We show that a pair of conserved proline residues in free chaperone forms a ‘proline lock’, which blocks the usher binding. Binding of subunit to the chaperone opens the proline lock and allows the chaperone-subunit complex to bind to the usher. We show that this proline lock exists in other CU systems and represents a general allosteric mechanism for selective targeting of chaperone:subunit complexes to the usher and for release and recycling of free chaperone. In paper I, a novel middle domain of Caf1A usher (UMD) was isolated and its crystal structure was determined. We show that UMD and Caf1 fibre subunit displayed significant structural similarity. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1-fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher