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

Abstract

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin α, with several human isoforms; among them, importin α3 (Impα3) 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 Impα3 and its depleted species, ∆Impα3, without 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 α3 with a low micromolar affinity (~5 μM). 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 ∆Impα3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Impα3, confirming that the IBB could act as an auto-inhibition region of Impα3. 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.

Influence of the disordered domain structure of MeCP2 on its structural stability and dsDNA interaction

“…One explanation could be that in experimental assays where the steady-state effect of the interaction between protein and ligand is observed after long incubation (e.g., steady-state spectroscopy, chemical and thermal denaturations, inhibition assays) there is sufficient time for the complex to achieve the optimal binding configuration, whereas in experimental assays where the transient effect of the interaction in the first minutes after instantaneous mixing (e.g., ITC) there might not be sufficient time to achieve optimal final binding configuration and lower binding affinities may be estimated. In this sense, differences in binding affinities as determined by different techniques or a slow adaptation of the ligand within the binding site to achieve an optimal binding affinity have been observed before for biological systems [43][44][45]. However, steady-state fluorescence spectroscopy experiments provided binding affinities for methylated and unmethylated dsDNA similar to those determined by ITC (Fig.…”

Human Enzyme PADI4 Binds to the Nuclear Carrier Importin α3

“…PADI4, Impα3, and ΔImpα3 were purified as previously described [ 24 , 41 , 42 , 43 ]. The concentrations of the proteins were calculated by UV absorbance, using an extinction coefficient at 280 nm; this parameter was estimated from the number of tyrosines and tryptophans in each of these proteins [ 44 ].…”

“…Two-dimensional spectra of NLS2-PADI4 (at 1.2 mM) were acquired in each dimension in phase-sensitive mode by using the time-proportional phase incrementation technique [ 55 ] and a spectral width of 5500 Hz. Standard TOCSY (mixing time of 80 ms) [ 56 ] and NOESY experiments (a mixing time of 250 ms) [ 56 , 57 , 58 ], with the WATERGATE sequence [ 53 ], as well as experimental, processing, and assigning details, were the same used in acquiring, processing, and analyzing the spectra of other NLSs [ 41 , 42 , 43 ]. The chemical shift values of H α protons in random-coil regions were obtained from tabulated data, corrected by neighboring residue effects [ 59 , 60 , 61 ].…”

“…We considered Impα3 as a target for PADI4 because (i) it is largely conserved among different species [ 39 ], and (ii) it has increased flexibility compared with other importins, as concluded by the structural B-factors from X-ray data; this feature confers this importin isoform a greater ability to interact with various cargos [ 40 ]. From an experimental point of view, Impα3 can also be easily expressed and purified for in vitro structural and binding studies [ 40 , 41 , 42 ]. In addition, Impα3 can be considered a model protein to investigate how the NLS sequence of the cargo can affect the thermodynamic parameters in the binding process, and we have already carried out several studies of the binding of Impα3 with other NLSs which can be used as a comparison [ 41 , 42 , 43 ].…”

“…From an experimental point of view, Impα3 can also be easily expressed and purified for in vitro structural and binding studies [ 40 , 41 , 42 ]. In addition, Impα3 can be considered a model protein to investigate how the NLS sequence of the cargo can affect the thermodynamic parameters in the binding process, and we have already carried out several studies of the binding of Impα3 with other NLSs which can be used as a comparison [ 41 , 42 , 43 ]. Lastly, by studying both importin species (with and without the IBB), we could explore whether the absence of the IBB domain affects the binding of the peptide encompassing the NLS region, as studied in the case of other NLSs of several proteins (see [ 41 , 42 , 43 ] and references therein).…”