Fine-tuned interactions between globular and disordered regions of single-stranded DNA binding (SSB) protein are required for dynamic condensation under physiological conditions.

Increasing evidence points to the importance of liquid-liquid phase separation (LLPS)-driven protein condensation in both eukaryotic and bacterial cell physiology. The formation of condensates may involve interactions between both structured (globular) domains and intrinsically disordered protein regions and requires multivalency that is often brought about by oligomerization. Here we dissect such contributions by assessing engineered variants of bacterial (Escherichia coli) single-stranded DNA binding (SSB) protein whose condensation has recently been implicated in bacterial genome metabolism. A truncated SSB variant (SSBdC, lacking the conserved C-terminal peptide (CTP)) was used to assess the importance of interactions between SSB's globular oligonucleotide/oligosaccharide binding (OB) domain and the CTP. We show that OB-CTP interactions are essential for dynamic condensation in physiological (crowded, glutamate-rich) environments. Via assessment of a protein variant (SSB^H55Y) from the known thermosensitive ssb-1 mutant, we also show that the perturbation of OB-OB contacts significantly impairs the stability of SSB tetramers and results in thermally induced protein aggregation, underscoring the importance of multivalence brought about by stereospecific contacts. Our data point to adaptive fine-tuning of SSB interactions to physiological condensation and demonstrate that SSB represents a versatile system for selective engineering of condensation-driving interactions between globular and disordered regions.