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Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody

PglK is an ABC transporter that flips a lipid-linked oligosaccharide (LLO) that serves as a donor in protein N-glycosylation. Previous structures revealed two inward-facing conformations, both with very large separations of the nucleotide binding domains (NBDs), and a closed, ADP-bound state that fe...

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Autores principales: Perez, Camilo, Köhler, Martin, Janser, Daniel, Pardon, Els, Steyaert, Jan, Zenobi, Renato, Locher, Kaspar P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395944/
https://www.ncbi.nlm.nih.gov/pubmed/28422165
http://dx.doi.org/10.1038/srep46641
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author Perez, Camilo
Köhler, Martin
Janser, Daniel
Pardon, Els
Steyaert, Jan
Zenobi, Renato
Locher, Kaspar P.
author_facet Perez, Camilo
Köhler, Martin
Janser, Daniel
Pardon, Els
Steyaert, Jan
Zenobi, Renato
Locher, Kaspar P.
author_sort Perez, Camilo
collection PubMed
description PglK is an ABC transporter that flips a lipid-linked oligosaccharide (LLO) that serves as a donor in protein N-glycosylation. Previous structures revealed two inward-facing conformations, both with very large separations of the nucleotide binding domains (NBDs), and a closed, ADP-bound state that featured an occluded cavity. To investigate additional states, we developed conformation-sensitive, single-domain camelid nanobodies (Nb) and studied their effect on PglK activity. Biochemical, structural, and mass spectrometric analyses revealed that one inhibitory Nb binds as a single copy to homodimeric PglK. The co-crystal structure of this Nb and ADP-bound PglK revealed a new, narrowly inward-open conformation. Rather than inducing asymmetry in the PglK homodimer, the binding of one Nb results in steric constraints that prevent a second Nb to access the symmetry-related site in PglK. The Nb performed its inhibitory role by a “sticky-doorstop” mechanism, where inhibition of ATP hydrolysis and LLO flipping activity occurs due to impaired closing of the NBD interface, which prevents PglK from converting to an outward-open conformation. This inhibitory mode suggests tight conformational coupling between the ATPase sites, which may apply to other ABC transporters.
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spelling pubmed-53959442017-04-21 Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody Perez, Camilo Köhler, Martin Janser, Daniel Pardon, Els Steyaert, Jan Zenobi, Renato Locher, Kaspar P. Sci Rep Article PglK is an ABC transporter that flips a lipid-linked oligosaccharide (LLO) that serves as a donor in protein N-glycosylation. Previous structures revealed two inward-facing conformations, both with very large separations of the nucleotide binding domains (NBDs), and a closed, ADP-bound state that featured an occluded cavity. To investigate additional states, we developed conformation-sensitive, single-domain camelid nanobodies (Nb) and studied their effect on PglK activity. Biochemical, structural, and mass spectrometric analyses revealed that one inhibitory Nb binds as a single copy to homodimeric PglK. The co-crystal structure of this Nb and ADP-bound PglK revealed a new, narrowly inward-open conformation. Rather than inducing asymmetry in the PglK homodimer, the binding of one Nb results in steric constraints that prevent a second Nb to access the symmetry-related site in PglK. The Nb performed its inhibitory role by a “sticky-doorstop” mechanism, where inhibition of ATP hydrolysis and LLO flipping activity occurs due to impaired closing of the NBD interface, which prevents PglK from converting to an outward-open conformation. This inhibitory mode suggests tight conformational coupling between the ATPase sites, which may apply to other ABC transporters. Nature Publishing Group 2017-04-19 /pmc/articles/PMC5395944/ /pubmed/28422165 http://dx.doi.org/10.1038/srep46641 Text en Copyright © 2017, 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
Perez, Camilo
Köhler, Martin
Janser, Daniel
Pardon, Els
Steyaert, Jan
Zenobi, Renato
Locher, Kaspar P.
Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody
title Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody
title_full Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody
title_fullStr Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody
title_full_unstemmed Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody
title_short Structural basis of inhibition of lipid-linked oligosaccharide flippase PglK by a conformational nanobody
title_sort structural basis of inhibition of lipid-linked oligosaccharide flippase pglk by a conformational nanobody
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395944/
https://www.ncbi.nlm.nih.gov/pubmed/28422165
http://dx.doi.org/10.1038/srep46641
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