Discrimination against RNA Backbones by a ssDNA Binding Protein

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Pot1 is a key shelterin component safeguarding telomeric single-stranded DNA (ssDNA) overhangs in eukaryotes.
• The Pot1 protein C-terminal domain (Pot1pC) displays non-specific ssDNA recognition via flexible binding conformations.
• The mechanism for Pot1pC's essential ssDNA over RNA specificity remains unclear despite its flexible binding.

Purpose of the Study:

• To elucidate how Pot1pC achieves specificity for ssDNA over RNA.
• To investigate the structural basis of Pot1pC's discrimination against RNA ligands.

Main Methods:

• Analysis of Pot1pC's ribose-position specificity with ssDNA and RNA-DNA chimeric ligands.
• Determination of high-resolution crystal structures of Pot1pC complexes with chimeric ligands.
• Examination of protein and ligand flexibility in binding interactions.

Main Results:

• Pot1pC exhibits additive, but not uniform, specificity across the ssDNA ligand.
• High-resolution structures reveal Pot1pC utilizes non-compensatory binding modes to discriminate against RNA.
• Significant binding interface rearrangement and alternative conformations occur, recovering most but not all binding energy.

Conclusions:

• Pot1pC achieves ssDNA over RNA specificity through adaptive binding interface rearrangements.
• Protein and ligand flexibility are crucial for accessing alternative conformations and tuning binding affinity.
• Intermolecular interfaces demonstrate sophisticated mechanisms for achieving specificity with flexible ligands.