Are the intrinsically disordered linkers involved in SSB binding to accessory proteins?

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Escherichia coli single-strand DNA binding (SSB) protein is crucial for genome maintenance, protecting ssDNA and interacting with numerous SSB-interacting proteins (SIPs).
• The C-termini of SSB protein feature an acidic tip and an intrinsically disordered linker (IDL), with the acidic tip known to interact with SIPs.
• A recent hypothesis suggests the IDL may also contribute to SIP interactions, prompting further investigation into its role.

Purpose of the Study:

• To investigate the binding interactions between SSB peptides, varying in IDL length, and four specific SIPs: RecO, PriC, PriA, and the χ subunit of DNA polymerase III.
• To determine whether the intrinsically disordered linker (IDL) of the SSB protein contributes to SIP binding affinity and specificity.
• To elucidate the binding characteristics of RecO to both SSB peptides and the intact SSB tetramer/ssDNA complex.

Main Methods:

• Synthesized three peptides comprising the SSB acidic tip with varying lengths of the intrinsically disordered linker (IDL).
• Utilized biophysical techniques to examine the binding affinities of these peptides to RecO, PriC, PriA, and the χ subunit of DNA polymerase III.
• Performed thermodynamic analyses of RecO binding to the SSB tetramer and an SSB-ssDNA complex, comparing it to peptide binding.

Main Results:

• Peptide binding affinities to individual SIPs were independent of IDL length, indicating that the acidic tip alone mediates these interactions.
• The SSB acidic tip demonstrated specificity, with binding affinities decreasing in the order: RecO > PriA ≈ χ > PriC.
• RecO binding to the SSB tetramer and SSB-ssDNA complex showed significant thermodynamic differences compared to peptide binding, suggesting additional interactions beyond the IDL.

Conclusions:

• The intrinsically disordered linker (IDL) of the SSB protein does not appear to contribute to the binding of the studied SIPs.
• The acidic tip of SSB is the primary determinant for interactions with RecO, PriC, PriA, and the χ subunit, exhibiting specific binding preferences.
• RecO likely interacts with other regions of the SSB protein, not just the C-terminal peptides, especially when bound to ssDNA or as a tetramer.