A conserved stable core structure in the passenger domain β‐helix of autotransporter virulence proteins

Abstract

In Gram‐negative bacteria, a wide variety of virulence factors are secreted via the autotransporter (AT) pathway. Intriguingly, there is no significant concentration of ATP in the periplasm, nor a proton gradient across the OM, so the energetic origin of efficient secretion of AT proteins is unknown. More than 97% of AT proteins are predicted to contain right‐handed parallel β‐helical structure, and the three crystal structures available for AT passenger domains each contain a long right‐handed parallel β‐helix. Previous studies have shown that pertactin, an AT from Bordetella pertussis, exhibits three‐state folding and has a C‐terminal stable core structure. Here, we show that Pet, an unrelated AT from Escherichia coli, also exhibits three‐state unfolding and also has a stable core structure. Deletion mutants, mass spectrometry, and N‐terminal sequencing demonstrate that the Pet stable core is also located near the C‐terminus of the passenger domain. Moreover, sequence analysis suggests that three‐state folding and a C‐terminal stable core structure could be important general features of the biogenesis of AT proteins in vivo. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 420–427, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

“…2) The rate of folding is expected to be relatively slow. The rate of folding of an AT passenger in vitro is extremely slow (k ϳ 10 Ϫ4 s Ϫ1 for pertactin (30)) and can take days to reach equilibrium (8,33). Folding in vivo obviously needs to be faster than that, but given the large size and high contact order of most AT passenger domains, we estimated the rate constant at ϳ10 Ϫ3 s Ϫ1 .…”

Interactions between EB1 and Microtubules

“…To test this idea, we performed proteinase K digestion assays. It is well established that proteinase K digestions provide a sensitive assay for differences in protein structure (41,42). The expectation is that if the His-tag (EB1 1-248 ) lines.…”

Crystal Structure of the Autochaperone Region from the Shigella flexneri Autotransporter IcsA

“…Limited proteolysis experiments of chemically denatured pertactin (12) and plasmid-encoded toxin from E. coli (26) showed the presence of a stable core at the C termini of these passenger domains. To characterize the stability of IcsA-AC, we used far-UV circular dichroism (CD) spectroscopy.…”