The plug domain of FepA, a TonB-dependent transport protein from Escherichia coli, binds its siderophore in the absence of the transmembrane barrel domain

Ken C. Usher, Engin Özkan, Kevin H. Gardner, Johann Deisenhofer

Research output: Contribution to journalArticle

51 Scopus citations

Abstract

FepA, an outer membrane iron siderophore transporter from Escherichia coli, is composed of a 22-stranded membrane-spanning β barrel with a globular N-terminal "plug" domain of 148 residues that folds up inside the barrel and completely occludes the barrel's interior (1). We have overexpressed and purified this plug domain by itself and find that it behaves in vitro as a predominantly unfolded yet soluble protein, as determined by circular dichroism, thermal denaturation, and NMR studies. Despite its unfolded state, the isolated domain binds ferric enterobactin, the siderophore ligand of FepA, with an affinity of 5 μM, just 100-fold reduced from that of intact FepA. These findings argue against the hypothesis that the plug domain is pulled intact from the barrel during transport in vivo but may be consistent either with a model where the plug rearranges within the barrel to create a channel large enough to allow transport or with a model where the plug unfolds and comes out of the barrel.

Original languageEnglish (US)
Pages (from-to)10676-10681
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number19
DOIs
StatePublished - Sep 11 2001

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