Conformational Polymorphism in Conditionally Disordered Nucleophosmin: From Single-Molecules to Liquid Droplets

Abstract

The order-disorder transitions of many intrinsically disordered proteins (IDPs) are fundamental in their biological functions, and post-translational modifications (PTMs) and partner binding have been proposed to act as molecular switches to regulate these transitions. The N-terminal oligomerization domain of the multi-functional nucleolar protein, nucleophosmin (NPM1; Npm-N), belongs to this emerging class of “conditionally” disordered proteins. Npm-N can interconvert between a globally disordered monomer and a β-sheet enriched pentamer forms which are differentially populated in different cellular functional and localization states of the protein, and can be tuned by phosphorylation and binding partners. However, the mechanism of this modulation remains poorly understood. Here, using a combination of single-molecule and ensemble experiments, we analyzed the disorder-order transition pathways of Npm-N and investigated how phospho-modifications and partner binding alter its folding landscape. Strikingly, we observed here that under conditions that favored forward (pentamer formation) vs. reverse (dissociation to monomers) reactions, order-disorder transitions in this single system can occur via either folding-induced-assembly or assembly-induced-folding, the two broad mechanistic classes for IDP coupled folding and binding. Furthermore, phosphorylation in Npm-N caused pathway dependent asymmetric effects in temporally decoupling the folding and assembly, thereby increasing the life-times of the intermediate states in these multi-step reaction pathways. Conversely, binding to arginine-rich motifs of the Arf tumor suppressor locked Npm-N in the ordered pentameric state, and could counteract the effects of phosphorylation. Finally, single-molecule along with ensemble experiments directly revealed substantial conformational alterations in Npm-N upon transition to liquid-droplets mediated by partner binding, a phenomenon with relevance to structure and function of the nucleolus. Our findings provide novel mechanistic insights into the complex interplay of the conformational biophysics and different cellular states, functions and regulation of this multi-functional protein.