Directed epitope delivery across the Escherichia coli outer membrane through the porin OmpF

Abstract

The porins OmpF and OmpC are trimeric β-barrel proteins with narrow channels running through each monomer that exclude molecules >600 Da while mediating the passive diffusion of small nutrients and metabolites across the Gram-negative outer membrane (OM). Here, we elucidate the mechanism by which an entire soluble protein domain (>6 kDa) is delivered through the lumen of such porins. Following high-affinity binding to the vitamin B 12 receptor in Escherichia coli, the bacteriocin ColE9 recruits OmpF or OmpC using an 83-residue intrinsically unstructured translocation domain (IUTD) to deliver a 16-residue TolB-binding epitope (TBE) in the center of the IUTD to the periplasm where it triggers toxin entry. We demonstrate that the IUTD houses two OmpFbinding sites, OBS1 (residues 2-18) and OBS2 (residues 54-63), which flank the TBE and bind with K d s of 2 and 24 μM, respectively, at pH 6.5 and 25 ºC. We show the two OBSs share the same binding site on OmpF and that the colicin must house at least one of them for antibiotic activity. Finally, we report the structure of the OmpF-OBS1 complex that shows the colicin bound within the porin lumen spanning the membrane bilayer. Our study explains how colicins exploit porins to deliver epitope signals to the bacterial periplasm and, more broadly, how the inherent flexibility and narrow crosssectional area of an IUP domain can endow it with the ability to traverse a biological membrane via the constricted lumen of a β-barrel membrane protein.colicin translocation | intrinsically unstructured protein | protein-protein interaction | transmembrane signalling | isothermal titration calorimetry

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

“…The relative intensities of bound to unbound protein were extracted and plotted as a function of OBS1 concentration (Figure S3). The K d determined (0.7±0.34 μ m ) for this membrane protein complex is in agreement with values reported (1.0±0.1 μ m ) 24. That the solution state equilibria is maintained is surprising given that DESI involves ejection of the membrane protein complex, deposited on the surface, by a desorption spray.…”

“…Exploring further the quantitative aspects of this native DESI platform we selected OBS1 a 17‐residue peptide known to bind within the pores of OmpF with binding constants determined previously by both ITC and nanoES MS 24, 25. We deposited OmpF on the stage in OG detergent and incubated OmpF with increasing concentrations of OBS1 (0–75 μ m ) and deposited these protein‐peptide complexes onto the native DESI stage.…”

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

“…1B) facilitates a search in two dimensions, via lateral diffusion and a "fishing pole" mechanism, by which the highly unstructured N-terminal T domain finds a copy of its OmpF translocator (8,13). For colicins E3 and E9, segments of their T domains were shown to be bound inside the pore of OmpF (14)(15)(16), and their T domains have also been shown to occlude OmpF channels in planar lipid bilayer membranes (16,17). For colicin Ia, a pore-forming colicin, a second copy of its Cir receptor serves as its translocator (10,18).…”

Outer membrane lipoprotein biogenesis: Lol is not the end

“…It is likely that the presence of an unstructured region, rather than sequence specificity, is the requirement for formation RcsF/OMP complexes. Strikingly, a similar type of interaction was observed for the unstructured linker region of a colicin during its entry through the OmpF lumen [102,103]. Finally, LptE is absolutely required for the folding and assembly of LptD.…”