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Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The...

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Autores principales: Ploetz, Evelyn, Schuurman-Wolters, Gea K., Zijlstra, Niels, Jager, Amarins W., Griffith, Douglas A., Guskov, Albert, Gouridis, Giorgos, Poolman, Bert, Cordes, Thorben
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025302/
https://www.ncbi.nlm.nih.gov/pubmed/33823661
http://dx.doi.org/10.1098/rsob.200406
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author Ploetz, Evelyn
Schuurman-Wolters, Gea K.
Zijlstra, Niels
Jager, Amarins W.
Griffith, Douglas A.
Guskov, Albert
Gouridis, Giorgos
Poolman, Bert
Cordes, Thorben
author_facet Ploetz, Evelyn
Schuurman-Wolters, Gea K.
Zijlstra, Niels
Jager, Amarins W.
Griffith, Douglas A.
Guskov, Albert
Gouridis, Giorgos
Poolman, Bert
Cordes, Thorben
author_sort Ploetz, Evelyn
collection PubMed
description The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.
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spelling pubmed-80253022021-04-16 Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ Ploetz, Evelyn Schuurman-Wolters, Gea K. Zijlstra, Niels Jager, Amarins W. Griffith, Douglas A. Guskov, Albert Gouridis, Giorgos Poolman, Bert Cordes, Thorben Open Biol Research The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously. The Royal Society 2021-04-07 /pmc/articles/PMC8025302/ /pubmed/33823661 http://dx.doi.org/10.1098/rsob.200406 Text en © 2021 The Authors. https://creativecommons.org/licenses/by/4.0/Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, provided the original author and source are credited.
spellingShingle Research
Ploetz, Evelyn
Schuurman-Wolters, Gea K.
Zijlstra, Niels
Jager, Amarins W.
Griffith, Douglas A.
Guskov, Albert
Gouridis, Giorgos
Poolman, Bert
Cordes, Thorben
Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ
title Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ
title_full Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ
title_fullStr Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ
title_full_unstemmed Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ
title_short Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ
title_sort structural and biophysical characterization of the tandem substrate-binding domains of the abc importer glnpq
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025302/
https://www.ncbi.nlm.nih.gov/pubmed/33823661
http://dx.doi.org/10.1098/rsob.200406
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