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A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-s...

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Autores principales: Neira, José L., Rizzuti, Bruno, Jiménez-Alesanco, Ana, Palomino-Schätzlein, Martina, Abián, Olga, Velázquez-Campoy, Adrián, Iovanna, Juan L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565984/
https://www.ncbi.nlm.nih.gov/pubmed/32933064
http://dx.doi.org/10.3390/biom10091313
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author Neira, José L.
Rizzuti, Bruno
Jiménez-Alesanco, Ana
Palomino-Schätzlein, Martina
Abián, Olga
Velázquez-Campoy, Adrián
Iovanna, Juan L.
author_facet Neira, José L.
Rizzuti, Bruno
Jiménez-Alesanco, Ana
Palomino-Schätzlein, Martina
Abián, Olga
Velázquez-Campoy, Adrián
Iovanna, Juan L.
author_sort Neira, José L.
collection PubMed
description Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7–27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-(1)H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.
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spelling pubmed-75659842020-10-26 A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin Neira, José L. Rizzuti, Bruno Jiménez-Alesanco, Ana Palomino-Schätzlein, Martina Abián, Olga Velázquez-Campoy, Adrián Iovanna, Juan L. Biomolecules Article Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, of which there are several human isoforms; among them, importin α3 (Impα3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54–74) of NUPR1 towards Impα3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impα3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impα3 with low micromolar affinity (1.7–27 μM). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important; furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-(1)H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure. MDPI 2020-09-11 /pmc/articles/PMC7565984/ /pubmed/32933064 http://dx.doi.org/10.3390/biom10091313 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Neira, José L.
Rizzuti, Bruno
Jiménez-Alesanco, Ana
Palomino-Schätzlein, Martina
Abián, Olga
Velázquez-Campoy, Adrián
Iovanna, Juan L.
A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin
title A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin
title_full A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin
title_fullStr A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin
title_full_unstemmed A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin
title_short A Phosphorylation-Induced Switch in the Nuclear Localization Sequence of the Intrinsically Disordered NUPR1 Hampers Binding to Importin
title_sort phosphorylation-induced switch in the nuclear localization sequence of the intrinsically disordered nupr1 hampers binding to importin
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565984/
https://www.ncbi.nlm.nih.gov/pubmed/32933064
http://dx.doi.org/10.3390/biom10091313
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