Abstract
The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.
Highlights
intrinsically disordered protein (IDP) have a dynamic nature that cannot be described with a single conformation
The far-UV circular dichroism (CD) spectra indicate that, at increasing concentrations of any of the crowders, there were no major changes in the spectra and in the percentage of secondary structure as it could be estimated from CD (Figure 1)
These findings indicate that crowders, in the concentration range explored, did not appear to alter the disordered nature of NUPR1
Summary
IDPs have a dynamic nature that cannot be described with a single conformation. They are mainly involved in protein-protein and protein-nucleic acid interactions which modulate several biological processes, such as transcriptional regulation, cell cycle control, replication, differentiation and RNA processing (Xie et al, 2007; Gsponer et al, 2008; Babu et al, 2011; Uversky, 2013; Wright and Dyson, 2015; Berlow et al, 2018). IDPs are engaged in multivalent and/or promiscuous interactions; hub proteins involved in the nodes of interaction networks, modulating the signals of several protein pathways, have a large proportion of disorder in their sequences (Hu et al, 2017). In spite of their dynamic nature, IDPs are far from being fully disordered: they have significant structural heterogeneity (Konrat, 2015; Kurzbach et al, 2017), which could be modulated by changes in the molecular environment, binding to other macromolecules (normally, their natural partners), or by post-translational modification (PTM)—often, but not exclusively, in the form of phosphorylation (Iakoucheva et al, 2004). Different IDPs have been analysed in the presence of crowders (such as polyethylenglycol, Ficoll, and Dextran polymers) as these agents can simulate the crowding conditions in the internal spaces of prokaryotic and eukaryotic cells (Qu and Bolen, 2003; Roque et al, 2007; Breydo et al, 2014; Banks et al, 2018)
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