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Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD

Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bact...

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Autores principales: Mireku, S. A., Sauer, M. M., Glockshuber, R., Locher, K. P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662663/
https://www.ncbi.nlm.nih.gov/pubmed/29084999
http://dx.doi.org/10.1038/s41598-017-14512-8
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author Mireku, S. A.
Sauer, M. M.
Glockshuber, R.
Locher, K. P.
author_facet Mireku, S. A.
Sauer, M. M.
Glockshuber, R.
Locher, K. P.
author_sort Mireku, S. A.
collection PubMed
description Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10(−6) and 10(−9) M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 10(4) and 10(6) M(−1)s(−1). For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies.
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spelling pubmed-56626632017-11-08 Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD Mireku, S. A. Sauer, M. M. Glockshuber, R. Locher, K. P. Sci Rep Article Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10(−6) and 10(−9) M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 10(4) and 10(6) M(−1)s(−1). For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies. Nature Publishing Group UK 2017-10-30 /pmc/articles/PMC5662663/ /pubmed/29084999 http://dx.doi.org/10.1038/s41598-017-14512-8 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Mireku, S. A.
Sauer, M. M.
Glockshuber, R.
Locher, K. P.
Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD
title Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD
title_full Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD
title_fullStr Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD
title_full_unstemmed Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD
title_short Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD
title_sort structural basis of nanobody-mediated blocking of btuf, the cognate substrate-binding protein of the escherichia coli vitamin b12 transporter btucd
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662663/
https://www.ncbi.nlm.nih.gov/pubmed/29084999
http://dx.doi.org/10.1038/s41598-017-14512-8
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