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

Abstract

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-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 Impα3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.

The Paralogue of the Intrinsically Disordered Nuclear Protein 1 Has a Nuclear Localization Sequence that Binds to Human Importin α3

“…Unfortunately, we cannot draw any defined conclusion for the intact NUPR1L, as we could not measure the binding parameters by ITC (Section 2.1). Nevertheless, the result obtained is in agreement with previous findings of other NLS regions of well-folded proteins [40] or with those of the intact NUPR1 protein (1.4 µM for Impα3) [23] or peptides comprising the NLS region of NUPR1 [41]. The presence of the IBB (which contains a large quantity of lysine amino acids) always exerts an auto-inhibitory effect, and the domain hampers the anchoring of NLS-NUPR1L into the major NLS-binding region of Impα3.…”

“…As the isolated NLS regions of both NUPR1 and NUPR1L contain the key residues to attain binding to Impα3, and with the peptides, we could measure the binding by ITC, we shall focus our attention on the comparison between the affinities of the two paralogues for those measurements. Comparison of the values of Table 1 for NLS-NUPR1L with those of the NLS region of NUPR1 (1.7 µM for Impα3 and 0.95 µM for ∆Impα3 [41]) indicate that the binding is stronger in the case of NUPR1. Therefore, although both paralogues bind to the same molecules ( [14] and this work), their affinity for the different partners is dissimilar.…”

“…In the case of the intact proteins, although we do not have the whole set of values of K d obtained with the same technique (for NUPR1L, the dissociation constants were obtained by fluorescence (Section 2.1), whereas for NUPR1, they were obtained by ITC [23,41]), it is important to consider that NUPR1L in solution is an oligomer and, therefore, the self-association equilibrium will affect the apparent values of the dissociation constants determined from the experiments, which could vary depending on the self-association state of the protein.…”

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

“…It is also implicated in apoptosis through the formation of a complex with prothymosin a ( Malicet et al, 2006 ), as well as intervening in DNA binding and repair ( Encinar et al, 2001 ; Aguado-Llera et al, 2013 ), and in the interaction with Polycomb group proteins ( Santofimia-Castaño et al, 2017 ). Moreover, NUPR1 also interacts with proteins involved in the nuclear transportation machinery, where phosphorylation at residue Thr68 modulates its transport into the nucleus driven by a helical conformational switch ( Lan et al, 2020 ; Neira et al, 2020 ). As PTMs can alter the local structure of NUPR1 (but not its overall disordered nature), we wondered whether changes in the solution conditions (in particular, in a crowded environment) could alter its flexibility and/or its local conformation.…”

The Amazing World of IDPs in Human Diseases

“…The interaction between the intrinsically disordered NUPR1 and the human importinα3 (Impα3) was investigated in depth by Neira et al [ 8 ] using several spectroscopic and biophysical techniques, including NMR and molecular docking. The authors focused on the affinity of the Nuclear Localization Sequence (NLS) of NUPR1 towards Impα3, taking into account several mutants of the NLS region, and demonstrated that the phosphorylation of Thr68 induces a conformational switch in the NLS region of NUPR1 which hampers binding to Impα3.…”