Thermodynamic Models for Assembly of Intrinsically Disordered Protein Hubs with Multiple Interaction Partners.

Prevalent in diverse protein interactomes, intrinsically disordered proteins or regions (IDPs or IDRs) often drive assembly of higher-order macromolecular complexes, using multiple target-binding motifs. Such IDP hubs are suggested to process various cellular signals and coordinate relevant biological processes. However, the mechanism of assembly and functional regulation of IDP hubs remains elusive due to the challenges in dissecting their intricate protein-protein interaction networks. Here we present basic thermodynamic models for the assembly of simple IDP hubs with multiple target proteins, constructing partition functions from fundamental binding parameters. We combined these basic functions to develop advanced thermodynamic models to analyze the assembly of the Nup153 hubs interacting with multiple karyopherin β1 (Kap) molecules, critical components of nucleocytoplasmic transport. The thermodynamic analysis revealed a complex organization of the Kap binding sites within the C-terminal IDR of Nup153 including a high-affinity 1:1 interaction site and a series of low-affinity sites for binding of multiple Kaps with negative cooperativity. The negative cooperativity arises from the overlapping nature of the low-affinity sites where Kap occupies multiple dipeptide motifs. The quantitative dissection culminated in construction of the Nup153 hub ensemble, elucidating how distribution among various Kap-bound states is modulated by Kap concentration and competing nuclear proteins. In particular, the Kap occupancy of the IDR can be fine-tuned by varying the location of competition within the overlapping sites, suggesting coupling of specific nuclear processes to distinct transport activities. In general, our results demonstrate the feasibility and a potential mechanism for manifold regulation of IDP functions by diverse cellular signals.