How Glutamate Promotes Liquid-liquid Phase Separation and DNA Binding Cooperativity of E. coli SSB Protein

• Published in: Journal of molecular biology 2022-05, Vol.434 (9), p.167562-167562, Article 167562
• Main Authors: Kozlov, Alexander G., Cheng, Xian, Zhang, Hongshan, Shinn, Min Kyung, Weiland, Elizabeth, Nguyen, Binh, Shkel, Irina A., Zytkiewicz, Emily, Finkelstein, Ilya J., Record, M. Thomas, Lohman, Timothy M.
• Format: Article
• Language: English
• Subjects: Amides - chemistry; biomolecular condensates; DNA replication; DNA, Single-Stranded - chemistry; DNA-Binding Proteins - chemistry; Escherichia coli Proteins - chemistry; Glutamic Acid - chemistry; Liquid-liquid phase separation; Phase Transition; Protein Binding; salt effects; single molecule DNA collapse
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2022.167562

[Display omitted] •Liquid-liquid phase separation of E. coli SSB protein is promoted by KGlu, but inhibited by KCl.•KGlu promotes NNN cooperativity and collapse of single molecules of SSB-coated polymeric ssDNA.•SSB LLPS and NNN cooperativity both require its intrinsically disordered linker (IDL) within its C-terminal tails.•KGlu interacts unfavorably with amide groups within the SSB IDL, whereas KCl interacts favorably.•ssDNA binding inhibits SSB phase separation. E. coli single-stranded-DNA binding protein (EcSSB) displays nearest-neighbor (NN) and non-nearest-neighbor (NNN)) cooperativity in binding ssDNA during genome maintenance. NNN cooperativity requires the intrinsically-disordered linkers (IDL) of the C-terminal tails. Potassium glutamate (KGlu), the primary E. coli salt, promotes NNN-cooperativity, while KCl inhibits it. We find that KGlu promotes compaction of a single polymeric SSB-coated ssDNA beyond what occurs in KCl, indicating a link of compaction to NNN-cooperativity. EcSSB also undergoes liquid–liquid phase separation (LLPS), inhibited by ssDNA binding. We find that LLPS, like NNN-cooperativity, is promoted by increasing [KGlu] in the physiological range, while increasing [KCl] and/or deletion of the IDL eliminate LLPS, indicating similar interactions in both processes. From quantitative determinations of interactions of KGlu and KCl with protein model compounds, we deduce that the opposing effects of KGlu and KCl on SSB LLPS and cooperativity arise from their opposite interactions with amide groups. KGlu interacts unfavorably with the backbone (especially Gly) and side chain amide groups of the IDL, promoting amide-amide interactions in LLPS and NNN-cooperativity. By contrast, KCl interacts favorably with these amide groups and therefore inhibits LLPS and NNN-cooperativity. These results highlight the importance of salt interactions in regulating the propensity of proteins to undergo LLPS.