Interactions of intrinsically disordered proteins

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Abstract

It has been estimated that approximately one third of all amino acids in proteins are in disordered chains, including disordered protein regions or fully disordered proteins. Their dynamic nature enhances the potential for behavior such as liquid-liquid phase separation (the formation of molecular condensates and membrane-less organelles), molecular switches through transient folding or post-translational modifications, dynamic intramolecular loops, and high sequence variability. Disordered proteins are particularly prevalent in RNA virus proteins. The nucleocapsid protein of SARS-CoV-2 (N-protein) presents a unique model system as a protein with three large disordered regions, due to the availability of the complete mutational landscape and data on molecular evolution. We have previously demonstrated how critical aspects of N-protein functions are enabled by the presence of intrinsic disorder: 1) the ability of flexible chains to accommodate large sequence variations to display a wide range of short linear motifs for binding host proteins; 2) the key transient folding motif in the central disordered linker to generate weak, promiscuous coiled-coils as a starting point for ribonucleoprotein particle formation in viral assembly; and 3) the non-local effect of point mutations in the flexible disordered regions generating a large biophysical parameter space for enhanced evolvability. In the review period we have explored two additional features that rest on the intrinsic disorder of N-protein: the formation of intra-molecular loops that can be switched by post-translational modifications, thus supporting distinct N-protein functions; and the ability to form polydisperse and pleomorphic complexes with RNA to enhance evolvability. The latter overlaps with our projects on the formation of ribonucleoprotein particles (RNPs) through N-protein interactions in viral assembly and is described in these reports. It has long been observed that N-protein in virions is hypophosphorylated, whereas intracellular N-protein is hyperphosphorylated in the SR-rich region of its central disordered linker. The functional consequence of phosphorylation is under active investigation by several laboratories, and phosphorylation inhibitors have been proposed as potential drug candidates. Based on recently published NMR chemical shift data, it has been proposed that the phosphorylated SR-rich disordered linker region (pSR) – as a result of its intrinsic flexibility – can fold back into the nucleic acid binding site of the adjacent folded nucleic acid binding domain (NTD), forming an intramolecular loop (pSR-loop). It was also suggested that different loops can form between the distant linker region and the NTD, in a way that is inhibitory for N-protein self-association in the leucine-rich sequence (LRS) of the linker (LRS-loop). We have set out to verify the existence and explore consequences of these mutually exclusive loops. To this end we have prepared unmodified and in-vitro enzymatically phosphorylated N-protein, as well as a phosphomimetic N-protein mutant. Consistent with the formation of the pSR-loop, we observed significant hydrodynamic compaction of the phosphorylated protein. In addition, we have discovered a significant impact of phosphorylation on N-protein reversible self-association, consistent with the release of the inhibitory LRS-loop. Furthermore, both loops impact the thermodynamic stability of the NTD domain, observed by differential scanning fluorimetry. Finally, we have developed an approach that will allow us to measure the population of the different loops under different conditions. This work contributes to the elucidation of the molecular switch of N-protein functions through phosphorylation, and aids in understanding consequences of potential phosphorylation inhibitors. At the same time, this study expands our methodological toolset for detecting and analyzing intramolecular loops of intrinsically disordered protein regions and their coupling to posttranslational modifications and protein self-association.