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Concerted loop motion triggers induced fit of FepA to ferric enterobactin

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 re...

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Autores principales: Smallwood, Chuck R., Jordan, Lorne, Trinh, Vy, Schuerch, Daniel W., Gala, Amparo, Hanson, Mathew, Shipelskiy, Yan, Majumdar, Aritri, Newton, Salete M.C., Klebba, Phillip E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076525/
https://www.ncbi.nlm.nih.gov/pubmed/24981231
http://dx.doi.org/10.1085/jgp.201311159
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author Smallwood, Chuck R.
Jordan, Lorne
Trinh, Vy
Schuerch, Daniel W.
Gala, Amparo
Hanson, Mathew
Shipelskiy, Yan
Majumdar, Aritri
Newton, Salete M.C.
Klebba, Phillip E.
author_facet Smallwood, Chuck R.
Jordan, Lorne
Trinh, Vy
Schuerch, Daniel W.
Gala, Amparo
Hanson, Mathew
Shipelskiy, Yan
Majumdar, Aritri
Newton, Salete M.C.
Klebba, Phillip E.
author_sort Smallwood, Chuck R.
collection PubMed
description 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.
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spelling pubmed-40765252015-01-01 Concerted loop motion triggers induced fit of FepA to ferric enterobactin Smallwood, Chuck R. Jordan, Lorne Trinh, Vy Schuerch, Daniel W. Gala, Amparo Hanson, Mathew Shipelskiy, Yan Majumdar, Aritri Newton, Salete M.C. Klebba, Phillip E. J Gen Physiol Research Articles 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. The Rockefeller University Press 2014-07 /pmc/articles/PMC4076525/ /pubmed/24981231 http://dx.doi.org/10.1085/jgp.201311159 Text en © 2014 Smallwood et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Research Articles
Smallwood, Chuck R.
Jordan, Lorne
Trinh, Vy
Schuerch, Daniel W.
Gala, Amparo
Hanson, Mathew
Shipelskiy, Yan
Majumdar, Aritri
Newton, Salete M.C.
Klebba, Phillip E.
Concerted loop motion triggers induced fit of FepA to ferric enterobactin
title Concerted loop motion triggers induced fit of FepA to ferric enterobactin
title_full Concerted loop motion triggers induced fit of FepA to ferric enterobactin
title_fullStr Concerted loop motion triggers induced fit of FepA to ferric enterobactin
title_full_unstemmed Concerted loop motion triggers induced fit of FepA to ferric enterobactin
title_short Concerted loop motion triggers induced fit of FepA to ferric enterobactin
title_sort concerted loop motion triggers induced fit of fepa to ferric enterobactin
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076525/
https://www.ncbi.nlm.nih.gov/pubmed/24981231
http://dx.doi.org/10.1085/jgp.201311159
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