Polyphosphate Discriminates Protein Conformational Ensembles More Efficiently than DNA Promoting Diverse Assembly and Maturation Behaviors

Disordered proteins and domains often assemble into condensates with polyanionic nucleic acids, primarily via charge complementarity, regulating numerous cellular functions. However, the assembly mechanisms associated with the other abundant and ubiquitous, anionic, stress-response regulating polymer, polyphosphate (polyP), is less understood. Here, we employ the intrinsically disordered DNA binding domain (DBD) of cytidine repressor (CytR) from E.coli to study the nature of assembly processes and their maturation with polyP and DNA. Wild-type CytR forms metastable liquid-like condensates with polyP and DNA, while undergoing liquid-to-solid transition in the former and solubilizing in the latter. On mutationally engineering the ensemble to exhibit more or less structure and dimensions than the WT, the assembly process with polyP is directed to either condensates with partial time-dependent solubilization or spontaneous aggregation, respectively. The timescales of structural changes in the condensates formed by the folded mutant with polyP span tens of minutes, indicative of slow dynamics and highlights the tussle between folding driven by intra-molecular interactions and assembly mediated by inter-molecular interactions. On the other hand, the CytR variants form only liquid-like but metastable droplets with DNA which solubilize within a few hours. Polyphosphate induces large secondary-structure changes with two of the mutants adopting polyproline II-like structures within droplets, while DNA has only minimal structural effects. Our findings reveal how polyphosphate is able to more efficiently discern conformational heterogeneity in the starting protein ensemble, its structure, and compactness, with broad implications in assembly mechanisms involving polyP and stress response in bacterial systems.