Colicin N Binds to the Periphery of Its Receptor and Translocator, Outer Membrane Protein F

Abstract

SummaryColicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport.

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

“…Moreover, the results confirm that LPS-OmpF complexes form with a range of stoichiometries within each sample and are stable under SDS/PAGE conditions without boiling (18). Moreover, these ladders resemble those observed in preparations of native OmpF from E. coli OM fractions if LPS is not intentionally removed (18).…”

“…OmpF trimers show identical structural and electrophysiological properties to those that folded in vivo in the presence of LPS in the OM (17,18).…”

“…A crystal structure of the omptin protease Pla from Yersinia pestis showed density consistent with LPS acyl chains, but no density for the polar part of the glycolipid was observed (21,22). Using the predictions from FhuA, we predicted likely LPS binding sites on the OmpF trimer and showed that these corresponded to changes in 2D crystal structure when the antibacterial toxin colicin N displaced tightly bound LPS from the OmpF trimer (18).…”

“…The addition of LPS (Fig. 1A) to LPS-free in vitro folded OmpF trimers (18) creates, on SDS/PAGE, a characteristic ladder of increasing molecular mass due to the slower mobility of LPS-bound OmpF (Fig. 1B) (8).…”

Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

“…This mode of secretion has been predicted for colicin A and colicin N (31,32). Colicin N is thought to bind the periphery of OmpF on the cell surface (33) and cross the outer membrane in an unfolded state, after which it forms a channel in the lipopolysaccharide (LPS) adjacent to OmpF (34). Future experiments will investigate the specific mechanism of MccPDI entry into susceptible E. coli.…”