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Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions

The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell env...

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Autores principales: Nair, Asha V., Singh, Himansha, Raturi, Sagar, Neuberger, Arthur, Tong, Zhen, Ding, Ning, Agboh, Kelvin, van Veen, Hendrik W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137110/
https://www.ncbi.nlm.nih.gov/pubmed/27917857
http://dx.doi.org/10.1038/srep38052
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author Nair, Asha V.
Singh, Himansha
Raturi, Sagar
Neuberger, Arthur
Tong, Zhen
Ding, Ning
Agboh, Kelvin
van Veen, Hendrik W.
author_facet Nair, Asha V.
Singh, Himansha
Raturi, Sagar
Neuberger, Arthur
Tong, Zhen
Ding, Ning
Agboh, Kelvin
van Veen, Hendrik W.
author_sort Nair, Asha V.
collection PubMed
description The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.
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spelling pubmed-51371102017-01-27 Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions Nair, Asha V. Singh, Himansha Raturi, Sagar Neuberger, Arthur Tong, Zhen Ding, Ning Agboh, Kelvin van Veen, Hendrik W. Sci Rep Article The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport. Nature Publishing Group 2016-12-05 /pmc/articles/PMC5137110/ /pubmed/27917857 http://dx.doi.org/10.1038/srep38052 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Nair, Asha V.
Singh, Himansha
Raturi, Sagar
Neuberger, Arthur
Tong, Zhen
Ding, Ning
Agboh, Kelvin
van Veen, Hendrik W.
Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions
title Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions
title_full Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions
title_fullStr Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions
title_full_unstemmed Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions
title_short Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions
title_sort relocation of active site carboxylates in major facilitator superfamily multidrug transporter lmrp reveals plasticity in proton interactions
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137110/
https://www.ncbi.nlm.nih.gov/pubmed/27917857
http://dx.doi.org/10.1038/srep38052
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