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 alpha, of which there are several human isoforms; among them, importin alpha 3 (Imp alpha 3) 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 Imp alpha 3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Imp alpha 3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Imp alpha 3 with low micromolar affinity (1.7-27 mu M). 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-H-1-NMR (two-dimensional-proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Imp alpha 3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.