A phosphorylation-induced switch in the nuclear localization sequence of the intrinsically disordered nupr1 hampers binding to importin

Neira, J.L. ; Rizzuti, B. ; Jiménez-Alesanco, A. (Universidad de Zaragoza) ; Palomino-Schätzlein, M. ; Abián, O. (Universidad de Zaragoza) ; Velázquez-Campoy, A. (Universidad de Zaragoza) ; Iovanna, J.L.

Resumen: Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin a, of which there are several human isoforms; among them, importin a3 (Impa3) 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 Impa3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Impa3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Impa3 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-1H-NMR (two-dimensional–proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Impa3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.

Idioma: Inglés

DOI: 10.3390/biom10091313

Año: 2020

Publicado en: Biomolecules 10, 9 (2020), 1313 [22 pp.]
ISSN: 2218-273X

Factor impacto JCR: 4.879 (2020)

Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 96 / 296 = 0.324 (2020) - Q2 - T1

Factor impacto SCIMAGO: 1.125 - Molecular Biology (Q2) - Biochemistry (Q2)

Financiación: info:eu-repo/grantAgreement/ES/DGA/B25-17R
Financiación: info:eu-repo/grantAgreement/ES/DGA/E45-17R
Financiación: info:eu-repo/grantAgreement/ES/ISCIII/CPII13-00017
Financiación: info:eu-repo/grantAgreement/ES/ISCIII-ERDF-ESF/PI15-00663-Investing in your future
Financiación: info:eu-repo/grantAgreement/ES/ISCIII-ERDF-ESF/PI18-00349-Investing in your future
Financiación: info:eu-repo/grantAgreement/ES/MCIU-AEI-FEDER/BFU2016-78232-P
Financiación: info:eu-repo/grantAgreement/ES/MCIU-AEI-FEDER/RTI2018-097991-B-I00

Tipo y forma: Artículo (Versión definitiva)

Área (Departamento): Área Bioquímica y Biolog.Mole. (Dpto. Bioq.Biolog.Mol. Celular)