Nonspecific Recognition Is Achieved in Pot1pC through the Use of Multiple Binding Modes

Abstract

Summary Pot1 is the protein responsible for binding to and protecting the 3’ single-stranded DNA (ssDNA) overhang at most eukaryotic telomeres. Here we present the crystal structure of one of the two OB-folds (Pot1pC) that make up the ssDNA-binding domain in S. pombe Pot1. Comparison with the homologous human domain reveals unexpected structural divergence in the mode of ligand binding that explains the differing ligand requirements between species. Despite the presence of apparently base-specific hydrogen bonds, Pot1pC is able to bind a wide range of ssDNA sequences with thermodynamic equivalence. To address how Pot1pC binds ssDNA with little to no specificity, multiple structures of Pot1pC bound to non-cognate ssDNA ligands were solved. These structures reveal that this promiscuity is implemented through new binding modes that thermodynamically compensate for base-substitutions through alternate stacking interactions and new H-bonding networks.

Tying up the Ends: Plasticity in the Recognition of Single-Stranded DNA at Telomeres

“…20,21,71 Structures of both the complete human DNA-binding domain (DBD) and the 2 OB folds that together comprise the S. pombe DBD have been solved. 72–74 hPOT1 adopts an elongated structure comprised of these 2 OB folds that are closely linked together by a short 9 amino acid linker such that the two domains functionally bind a 10-nt telomeric ssDNA ligand as one contiguous unit with an extensive domain/domain interface (Figure 3A). hOB1, the N-terminal OB fold, binds the first 6-nt (TTAGGG) with strong specificity, especially for nucleotides 2–5.…”

“…21,72–74 This includes an N-terminal DNA-binding domain (Pot1-DBD) composed of two OB-folds (Pot1pN and Pot1pC). 72–74,82 Biochemical experiments already suggest a difference in mechanism of action between the homologs. Pot1pN and Pot1pC can be separated and retain biochemical activity individually, in contrast to hOB1 and hOB2 which appear to function only as a tightly packed unit.…”

“…Pot1pN and Pot1pC can be separated and retain biochemical activity individually, in contrast to hOB1 and hOB2 which appear to function only as a tightly packed unit. 73,74,82–84 This is likely in part due to the expanded linker between Pot1pN and Pot1pC, composed of 25 proteolytically labile residues as opposed to only 5 disordered residues between hOB1 and hOB2. 74,84 Thus Pot1pN and Pot1pC appear to be flexibly tethered subdomains in contrast to the more tightly packed arrangement between hOB1 and hOB2.…”

The OB-fold domain 1 of human POT1 recognizes both telomeric and non-telomeric DNA motifs

“…Recent studies have revealed an unexpected capability of Pot1pC to recognize a variety of non-cognate ligands [36,37]. Surprisingly, the non-cognate ligands were recognized through hydrogen bond interactions that typically contribute to sequence specificity.…”

“…Surprisingly, the non-cognate ligands were recognized through hydrogen bond interactions that typically contribute to sequence specificity. With the flexibility of the system, the broad DNA specificity was derived from local reorientations of bases and/or side chains or by global conformational changes affecting both protein and DNA [36,37]. Whether the human POT1 protein exhibits a similar flexibility and is able to interact with non-telomeric ligands has not yet been formally investigated.…”