Evidence of ball-and-chain transport of ferric enterobactin through FepA.
The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane beta-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy- and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the beta-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal beta-barrel domain confirmed the feasibility of this mechanism.
Ma
Li
L
Department of Chemistry & Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Kaserer
Wallace
W
Annamalai
Rajasekeran
R
Scott
Daniel C
DC
Jin
Bo
B
Jiang
Xiaoxu
X
Xiao
Qiaobin
Q
Maymani
Hossein
H
Massis
Liliana Moura
LM
Ferreira
Luiz C S
LC
Newton
Salete M C
SM
Klebba
Phillip E
PE
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D003087:Colicins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004758:Enterobactin / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D019793:Fluorescein / Q000494:pharmacology; D008301:Maleimides / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D018272:Porins / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000502:physiology*; D017262:Siderophores / Q000737:chemistry / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003087:Colicins; D008301:Maleimides; D018272:Porins; D011956:Receptors, Cell Surface; D017262:Siderophores; C029489:enterobactin receptor; C043592:maleimide; D004758:Enterobactin; D003545:Cysteine; D019793:Fluorescein