Discrimination against RNA Backbones by a ssDNA Binding Protein

Pot1 is the shelterin component responsible for the protection of the single-stranded DNA (ssDNA) overhang at telomeres in nearly all eukaryotic organisms. The C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this flexibility, it is unclear how specificity for ssDNA over RNA, an activity required for biological function, is achieved. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of several Pot1pC complexes with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities. These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity toward flexible ligands. [Display omitted] •ssDNA-binding Pot1pC discriminates against ssRNA at the 5′ end of the ligand•Structural insight obtained into ribose specificity in the context of base tolerance•Alternative binding mode enforces suboptimal binding geometries for RNA•Universally employed alternative binding mode accommodates modifications Lloyd et al. determine the nucleotide-position specificity of the C-terminal domain of S. pombe Pot1 for ssDNA over ssRNA and report three ligand-bound structures that reveal RNA-chimeric ligands bind in a widely utilized non-compensatory binding mode that features significant rearrangement of the binding interface to achieve thermodynamically equivalent binding.