Structural basis of DNA binding to human YB-1 cold shock domain regulated by phosphorylation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Human Y-box binding protein 1 (YB-1) is a multifunctional protein overexpressed in various cancers.
• YB-1 utilizes its cold shock domain (CSD) to bind nucleic acids and regulate metabolism.
• The C-terminal extension of CSD and S102 phosphorylation are critical for YB-1 function, but their roles remain unclear.

Purpose of the Study:

• To elucidate the structural and functional roles of the YB-1 CSD C-terminal extension (CSDex) and S102 phosphorylation.
• To investigate how these modifications affect YB-1's DNA/RNA binding and regulatory capabilities.

Main Methods:

• X-ray crystallography to determine the structure of human YB-1 CSDex.
• Structural analysis of CSDex in complex with single-stranded DNA (ssDNA).
• DNA-binding assays to assess the impact of the C-terminal extension and phosphorylation on binding affinity.

Main Results:

• The C-terminal extension is a rigid structure that interacts with the CSD, not disordered as previously assumed.
• Deletion of the extension or S102 phosphorylation destabilizes YB-1, leading to partial unfolding.
• CSDex binds ssDNA, with the extension providing a unique binding site; phosphorylation significantly reduces ssDNA binding.

Conclusions:

• The C-terminal extension and S102 phosphorylation are key regulators of YB-1's DNA/RNA binding affinity.
• Phosphorylation-dependent modulation of CSDex binding affinity likely controls gene transcription and translation.