Intrinsically disordered proteins and proteins containing intrinsically disordered regions are highly abundant in the proteome of eukaryotes and are extensively involved in essential biological functions. More recently, their role in the organization of biomolecular condensates has become evident and along with their misregulation in several neurologic disorders. Currently, most studies involving these proteins are carried out in vitro and using purified proteins. Given that in cells, condensate‐forming proteins are exposed to high, millimolar concentrations of cellular metabolites, we aimed to reveal the interactions of cellular metabolites and a representative condensate‐forming protein. Here, using the arginine–glycine/arginine–glycine–glycine (RG/RGG)‐rich cold inducible RNA binding protein (CIRBP) as paradigm, we studied binding of the cellular metabolome to CIRBP. We found that most of the highly abundant cellular metabolites, except nucleotides, do not directly bind to CIRBP. ATP, ADP, and AMP as well as NAD⁺, NADH, NADP⁺, and NADPH directly interact with CIRBP, involving both the folded RNA‐recognition motif and the disordered RG/RGG region. ATP binding inhibited RNA‐driven phase separation of CIRBP. Thus, it might be beneficial to include cellular metabolites in in vitro liquid–liquid phase separation studies of RG/RGG and other condensate‐forming proteins in order to better mimic the cellular environment in the future.

Arginine-glycine(-glycine) (RG/RGG) regions are highly abundant in RNA-binding proteins and involved in numerous physiological processes. Aberrant liquid-liquid phase separation (LLPS) and stress granule (SGs) association of RG/RGG regions in the cytoplasm have been implicated in several neurodegenerative disorders. LLPS and SG association of these proteins is regulated by the interaction with nuclear import receptors, such as transportin-1 (TNPO1), and by post-translational arginine methylation. Strikingly, many RG/RGG proteins harbour potential phosphorylation sites within or close to their arginine methylated regions, indicating a regulatory role. Here, we studied the role of phosphorylation within RG/RGG regions on arginine methylation, TNPO1-binding and LLPS using the cold-inducible RNA-binding protein (CIRBP) as a paradigm. We show that the RG/RGG region of CIRBP is in vitro phosphorylated by serine-arginine protein kinase 1 (SRPK1), and discovered two novel phosphorylation sites in CIRBP. SRPK1-mediated phosphorylation of the CIRBP RG/RGG region impairs LLPS and binding to TNPO1 in vitro and interferes with SG association in cells. Furthermore, we uncovered that arginine methylation of the CIRBP RG/RGG region regulates in vitro phosphorylation by SRPK1. In conclusion, our findings indicate that LLPS and TNPO1-mediated chaperoning of RG/RGG proteins is regulated through an intricate interplay of post-translational modifications.

Solvent paramagnetic relaxation enhancement (sPRE) is a versatile nuclear magnetic resonance (NMR)-based method that allows characterization of the structure and dynamics of biomolecular systems through providing quantitative experimental information on solvent accessibility of NMR-active nuclei. Addition of soluble paramagnetic probes to the solution of a biomolecule leads to paramagnetic relaxation enhancement in a concentration-dependent manner. Here we review recent progress in the sPRE-based characterization of structural and dynamic properties of biomolecules and their complexes, and aim to deliver a comprehensive illustration of a growing number of applications of the method to various biological systems. We discuss the physical principles of sPRE measurements and provide an overview of available co-solute paramagnetic probes. We then explore how sPRE, in combination with complementary biophysical techniques, can further advance biomolecular structure determination, identification of interaction surfaces within protein complexes, and probing of conformational changes and low-population transient states, as well as deliver insights into weak, nonspecific, and transient interactions between proteins and co-solutes. In addition, we present examples of how the incorporation of solvent paramagnetic probes can improve the sensitivity of NMR experiments and discuss the prospects of applying sPRE to NMR metabolomics, drug discovery, and the study of intrinsically disordered proteins.

Integrative structure of a complete cytokine receptor in a nanodisc opens for mechanistic decoding of disorder-based biology.

Millions of people worldwide are affected by neurodegenerative diseases (NDs), and to date, no effective treatment has been reported. The hallmark of these diseases is the formation of pathological aggregates and fibrils in neural cells. Many studies have reported that catechins, polyphenolic compounds found in a variety of plants, can directly interact with amyloidogenic proteins, prevent the formation of toxic aggregates, and in turn play neuroprotective roles. Besides harboring amyloidogenic domains, several proteins involved in NDs possess arginine-glycine/arginine-glycine-glycine (RG/RGG) regions that contribute to the formation of protein condensates. Here, we aimed to assess whether epigallocatechin gallate (EGCG) can play a role in neuroprotection via direct interaction with such RG/RGG regions. We show that EGCG directly binds to the RG/RGG region of fused in sarcoma (FUS) and that arginine methylation enhances this interaction. Unexpectedly, we found that low micromolar amounts of EGCG were sufficient to restore RNA-dependent condensate formation of methylated FUS, whereas, in the absence of EGCG, no phase separation could be observed. Our data provide new mechanistic roles of EGCG in the regulation of phase separation of RG/RGG-containing proteins, which will promote understanding of the intricate function of EGCG in cells.