부산대학교 도서관

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin a, with several human isoforms; among them, importin [alpha]3 (Imp[alpha]3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Imp[alpha]3 and its depleted species, AImp[alpha]3, withou the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp [alpha]3 with a low micromolar affinity (~5 [micro]M). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to AImp[alpha]3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Imp[alpha]3, confirming that the IBB could act as an auto-inhibition region of Imp[alpha]3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation. Keywords: circular dichroism; fluorescence; importin; intrinsically disordered protein (IDP); isothermal titration calorimetry (ITC); molecular docking; nuclear magnetic resonance (NMR); paralogue; peptide
1. Introduction NUPR1 (UniProtKB O60356) is an 82-residue-long (8 kDa), monomeric intrinsically disordered protein (IDP) with a large content of basic residues [1,2]. It does not have a stable secondary [...]