Crystal structures of the OmpF porin: function in a colicin translocon

Abstract

The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 Å OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co‐crystallization of OmpF with the unfolded 83 residue glycine‐rich N‐terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 Å structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly‐glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno‐extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease

“…Although OmpC and OmpF show high-sequence homology, they do not show any extended regions of amino acid sequence with significant homology with TSHR A-subunit, which may readily explain the nature of the cross-reactive determinants. Structural studies have shown that OmpC and OmpF adopt b barrel structures, with the extracellular regions adopting loop conformations (41,42); it is possible that some of these conformations may mimic the TSAbs epitopes in the LRR region of TSHR A-subunit. However, the pore function of OmpC and OmpF is dependent on adopting a trimeric structure (50), which may multiply the accessibility of the cross-reactive determinants to TSHR.…”

“…3A, 3B). The b sheet topology of TSHR A-subunit bears some similarity to the porin b barrel, but because the majority of the porin barrel is embedded in the membrane, we did not investigate this further but focused our attention on the extracellular loops of the porins (41,42). Given that all five N-terminal TSHR residues that are critical for M22 binding are positively charged (R38, K58, R80, H105, and K129) (14), we examined the electrostatic potential surface of the extracellular loops of the porins for clues to cross-reactivity.…”

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).…”

Characterization of the motion of membrane proteins using high-speed atomic force microscopy

“…Outer membrane protein F (OmpF) is the major porin in the outer membrane of Escherichia coli, where it forms trimeric channels for the passage of water, ions, sugars, polar nutrients and waste up to 700 Da in weight 5,6 . Diffusion in the membrane is an important factor in the function of OmpF as it has to be widely distributed on the cell surface for phage recognition 7 to occur, and also has to form a transient translocon with vitamin B 12 receptor BtuB for colicin import 8,9 .…”