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

FepA, an outer membrane iron siderophore transporter from Escherichia coli, is composed of a 22-stranded membrane-spanning beta barrel with a globular N-terminal "plug" domain of 148 residues that folds up inside the barrel a nd 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 dich roism, thermal denaturation, and NMR studies. Despite its unfolded state, t he isolated domain binds ferric enterobactin, the siderophore ligand of Fep A, with an affinity of 5 muM, just 100-fold reduced from that of intact Fep A. These findings argue against the hypothesis that the plug domain is pull ed intact from the barrel during transport in vivo but may be consistent ei ther with a model where the plug rearranges within the barrel to create a c hannel large enough to allow transport or with a model where the plug unfol ds and comes out of the barrel.