Folding and Secretion of the E. coli Pet Autotransporter

In Gram-negative bacteria, a wide range of virulence proteins are secreted via the autotransporter (AT) pathway. AT pre-proteins are synthesized with an N-terminal signal sequence that facilitates transport across the inner membrane, and a C-terminal β-barrel domain that is required for the mature virulence factor to cross the outer membrane (OM), flanking the central passenger domain that forms the mature, secreted virulence factor. Intriguingly, there is no significant concentration of ATP in the periplasm, nor a proton gradient across the OM, and hence the energetic origin of efficient OM secretion of AT proteins is unknown. Yet more than 97% of AT proteins are predicted to contain right-handed parallel β-helical structure, and the four of the five crystal structures available for AT passenger domains each contain a long right-handed parallel β-helix. In vitro folding studies of the pertactin passenger domain show that pertactin exhibits three state folding and has a C-terminal stable core structure. Here we show that an unrelated AT passenger domain Pet, also exhibits three state unfolding and has a C-terminal stable core structure. Stable core structure in the β-helix of AT passenger domains could be important for efficient OM secretion of the passenger domain. To test the requirement of different domains of the Pet passenger domain a series of passenger domain deletion mutants were constructed.

These studies revealed that while a passenger domain consisting of only the C-terminal stable core is secreted efficiently, deletion of the stable core results in lower levels of secreted passenger domain, which is not correctly folded, suggesting the stable core plays a role in both the secretion efficiency and folding of the mature passenger domain. The Pet passenger domain consists of an N- terminal protease domain in addition to the β-helix domain. To investigate the role of the N-terminal globular domain of the Pet passenger, we replaced the Pet protease domain with dihydrofolate reductase (DHFR), a protein whose folding and stability are well characterized. The chimeric DHFR-Pet β-helix passenger domain is secreted across the OM and stabilization of DFHR by methotrexate reduces the level of secreted chimera. Moreover, we altered the stability of the DHFR domain by constructing a series of point mutations, and observed a linear correlation between DHFR stability and OM secretion efficiency. In contrast, mutations that destabilize the pertactin stable core result in lower levels of OM secretion, while a mutation that lowers stability of only the N-terminus of the pertactin Ì_å_-helix has no effect on OM secretion. These results suggests that while the stability of the C-terminal stable core provides a positive contribution to OM secretion, stabilizing more N-terminal portions of the AT passenger domain can be detrimental to efficient OM secretion. These results demonstrate for the first time that passenger domain stability plays a direct role in AT OM secretion efficiency.