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

DOI: 10.1073/pnas.1010780107 DOI Help
PMID: 21098297 PMID Help

Authors: N. G. Housden (University of York) , Justyna A. Wojdyla (University of York) , Justyna Korczynska (University of York) , I. Grishkovskaya (University of York) , N. Kirkpatrick (University of York) , Andrzej Marek Brzozowski (York Structural Biology Laboratory, Department of Chemistry, University of York, U.K.) , C. Kleanthous (University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences , VOL 107 (50) , PAGES 21412 - 21417

State: Published (Approved)
Published: December 2010

Open Access Open Access

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 B12 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 OmpF-binding sites, OBS1 (residues 2–18) and OBS2 (residues 54–63), which flank the TBE and bind with Kds 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 cross-sectional 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.

Journal Keywords: Cell; Colicins; Crystallography; X-Ray; Epitopes; Escherichia; Models; Molecular; Porins; Protein; Tertiary; Recombinant Fusion Proteins

Diamond Keywords: Bacteria

Subject Areas: Biology and Bio-materials

Instruments: I04-Macromolecular Crystallography

Added On: 05/06/2015 11:24


Discipline Tags:

Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)