Charge-dependent secretion of an intrinsically disordered protein via the autotransporter pathway

Abstract

Autotransporters are a large class of virulence proteins produced by Gram-negative bacteria. They contain an N-terminal extracellular ("passenger") domain that folds into a β-helical structure and a C-terminal β-barrel ("β") domain that anchors the protein to the outer membrane. Because the periplasm lacks ATP, the source of energy that drives passenger domain secretion is unknown. The prevailing model postulates that vectorial folding of the β-helix in the extracellular space facilitates unidirectional secretion of the passenger domain. In this study we used a chimeric protein composed of the 675-residue receptor-binding domain (RD) of the Bordetella pertussis adenylate cyclase toxin CyaA fused to the C terminus of the Escherichia coli O157:H7 autotransporter EspP to test this hypothesis. The RD is a highly acidic, repetitive polypeptide that is intrinsically disordered in the absence of calcium. Surprisingly, we found that the RD moiety was efficiently secreted when it remained in an unfolded conformation. Furthermore, we found that neutralizing or reversing the charge of acidic amino acid clusters stalled translocation in the vicinity of the altered residues. These results challenge the vectorial folding model and, together with the finding that naturally occurring passenger domains are predominantly acidic, provide evidence that a net negative charge plays a significant role in driving the translocation reaction.bacterial pathogenesis | membrane protein assembly | type Va secretion

Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

“…However, constructs used in that study preserved a significant portion (Ͼ100 residues) from the C terminus of the wild type passenger, including a segment shown previously to be important for passenger folding and OM translocation (9, 10, 39 -41). In addition, many studies have found the AT secretion process to be severely disrupted under conditions that induced passenger folding in the periplasm (10,24,42,43), consistent with our simulation results. On balance, it appears that AT OM translocation can be severely disrupted either by premature folding in the periplasm or by a disruption of folding at the cell surface, particularly in the C-terminal region of the passenger.…”

“…At first glance, these results may seem to contradict the recent finding that AT OM translocation is not significantly disrupted in EspP mutants with a largely disordered passenger (24). However, constructs used in that study preserved a significant portion (Ͼ100 residues) from the C terminus of the wild type passenger, including a segment shown previously to be important for passenger folding and OM translocation (9, 10, 39 -41).…”

“…Additionally, although there are limits to the size and bulkiness of prefolded passenger structural elements that can cross the OM without blocking translocation (20,21), some of these structures are surprisingly large when compared with the 1-2-nm diameter of the translocator pore (22,23). Moreover, recent reports have shown that the range of passenger sequences and folding properties compatible with OM translocation is broader than previously thought (24,25). These results have introduced a considerable level of complexity and confusion about what was once thought to be a fairly straightforward and self-sufficient protein secretion mechanism.…”

Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

“…Plasmids were maintained by the addition of ampicillin (100 g/ml). Antisera against EspP C-terminal and N-terminal peptides and the purified EspP passenger domain have been described previously (30,31). Purified recombinant tobacco etch virus (TEV) protease (hyperactive S219V mutant) was obtained from Susan Buchanan.…”

Digested disorder, Quarterly intrinsic disorder digest (October-November-December, 2013)

“…Kang'ethe and Bernstein 42 produced a chimeric protein with a highly acidic receptor-binding domain (RD), to explore the mechanism of secretion. They found that the RD moiety was secreted efficiently when unfolded, but changing the pH stalled translocation.…”