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Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

BACKGROUND: The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble p...

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Autores principales: Wolters, Justina C., Berntsson, Ronnie P-A., Gul, Nadia, Karasawa, Akira, Thunnissen, Andy-Mark W. H., Slotboom, Dirk-Jan, Poolman, Bert
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861598/
https://www.ncbi.nlm.nih.gov/pubmed/20454456
http://dx.doi.org/10.1371/journal.pone.0010361
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author Wolters, Justina C.
Berntsson, Ronnie P-A.
Gul, Nadia
Karasawa, Akira
Thunnissen, Andy-Mark W. H.
Slotboom, Dirk-Jan
Poolman, Bert
author_facet Wolters, Justina C.
Berntsson, Ronnie P-A.
Gul, Nadia
Karasawa, Akira
Thunnissen, Andy-Mark W. H.
Slotboom, Dirk-Jan
Poolman, Bert
author_sort Wolters, Justina C.
collection PubMed
description BACKGROUND: The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized. PRINCIPAL FINDINGS: The binding of glycine betaine to purified OpuA and OpuAC (K(D) = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher. CONCLUSIONS: Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.
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spelling pubmed-28615982010-05-07 Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA Wolters, Justina C. Berntsson, Ronnie P-A. Gul, Nadia Karasawa, Akira Thunnissen, Andy-Mark W. H. Slotboom, Dirk-Jan Poolman, Bert PLoS One Research Article BACKGROUND: The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized. PRINCIPAL FINDINGS: The binding of glycine betaine to purified OpuA and OpuAC (K(D) = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher. CONCLUSIONS: Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site. Public Library of Science 2010-04-29 /pmc/articles/PMC2861598/ /pubmed/20454456 http://dx.doi.org/10.1371/journal.pone.0010361 Text en Wolters et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Wolters, Justina C.
Berntsson, Ronnie P-A.
Gul, Nadia
Karasawa, Akira
Thunnissen, Andy-Mark W. H.
Slotboom, Dirk-Jan
Poolman, Bert
Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA
title Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA
title_full Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA
title_fullStr Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA
title_full_unstemmed Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA
title_short Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA
title_sort ligand binding and crystal structures of the substrate-binding domain of the abc transporter opua
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861598/
https://www.ncbi.nlm.nih.gov/pubmed/20454456
http://dx.doi.org/10.1371/journal.pone.0010361
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