title: Concerted loop motion triggers induced fit of FepA to ferric enterobactin.
abstract: Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles.
authors: The Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019.
authors: Chuck R
authors: CR
authors: Smallwood
authors: The Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506.
authors: Lorne
authors: L
authors: Jordan
authors: The Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019.
authors: Vy
authors: V
authors: Trinh
authors: The Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019.
authors: Daniel W
authors: DW
authors: Schuerch
authors: The Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019.
authors: Amparo
authors: A
authors: Gala
authors: The Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506.
authors: Mathew
authors: M
authors: Hanson
authors: Matthew
authors: M
authors: Hanson
authors: The Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506.
authors: Yan
authors: Y
authors: Shipelskiy
authors: The Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506.
authors: Aritri
authors: A
authors: Majumdar
authors: The Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506.
authors: Salete M C
authors: SM
authors: Newton
authors: The Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506 peklebba@ksu.edu.
authors: Phillip E
authors: PE
authors: Klebba
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004758:Enterobactin / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli; D009038:Motion; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D011956:Receptors, Cell Surface; C029489:enterobactin receptor; D004758:Enterobactin; C014554:ferrienterochelin