Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer

Abstract

Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

Passive receptor dissociation driven by porin threading establishes active colicin transport throughEscherichia coliOmpF

“…The new model shows conclusively that OBS1 has its N-terminus pointing towards the extracellular environment (Figure 3a) and in both models the conformation adopted by OBS1 is identical. This orientation agrees with recent electrophysiological data and live-cell imaging experiments all of which show OBS1 binding subunit 1 of OmpF from the periplasm (Housden et al, 2018;Lee et al, 2020). In contrast to OBS1, the hydrogen bond network that stabilises OBS2 within subunit 2 of OmpF primarily involves OBS2 backbone atoms and OmpF side-chains (Figure 3h), likely explained by the high glycine content within the OBS2 sequence (6/11 residues are glycine, compared to 6/16 for OBS1).…”

Membrane protein channels equipped with a cleavable linker for inducing catalysis inside nanocompartments

“…22 Another strategy, ''bioinspired'' by cellular membranes where numerous proteins mediate molecular transport across the membrane, hinges on incorporating proteins or small polypeptides into the polymersome membrane to achieve a desired permeability. 23 For example, channel forming proteins such as the outer membrane protein F (OmpF), enable the diffusion of molecules with a cut-off largely corresponding to the pore diameter, 24,25 while others, such as Ferrichrome outer membrane transporter (FhuA) or gramicidin, only allow diffusion of certain small molecules or ions. 26,27 A step further in developing stimuli-responsive membrane permeabilization of polymersomes was achieved by turning inserted membrane proteins into ''bio-valves'' or ''bio-locks'' by corresponding modifications of the protein.…”

Toxin import through the antibiotic efflux channel TolC

“…Examples of energised transfer include pyocins S2 and S5 in P. aeruginosa, where both toxins exploit the PMF to deliver a TonB box through their OM TBDT translocator prior to import 13,29 . Passive transfer of bacteriocin disorder epitopes includes those of colicins E2-E9 in E. coli, whereby contact with TolB in the periplasm is achieved by insertion of the disordered sequence at the N-termini of these toxins through the pores of the general porins OmpF or OmpC 16,30 . Our protease protection assays suggest that KlebC similarly manages to passively translocate its disordered N-terminal domain, containing the TonB box, through the iris of TolC into the periplasm.…”