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 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 microM, 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.
Usher
K C
KC
Howard Hughes Medical Institute and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Ozkan
E
E
Gardner
K H
KH
Deisenhofer
J
J
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface; D017262:Siderophores / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D017262:Siderophores; C029489:enterobactin receptor; C057616:tonB protein, Bacteria; C489181:tonB protein, E coli