beta-hairpin peptides and WW domains designed for selective recognition of oligonucleotides.

摘要

Protein-nucleic acid interactions are crucial in a variety of biological processes. Protein interactions with single-stranded DNA are particularly important in DNA replication, repair, and telomere regulation. The interactions involved in the binding of a designed beta-hairpin dimer, (WKWK)2, to ssDNA and dsDNA were previously explored, and the peptide was found to bind ssDNA with a Kd of 3 muM via a combination of aromatic and electrostatic interactions, whereas binding to duplex DNA was driven primarily by electrostatic interactions. In this work, the effects of folding and chirality were studied to determine factors that contribute to affinity and selectivity for ssDNA versus dsDNA. Binding studies showed that (1) folding is crucial for binding to both ss- and dsDNA; and (2) chirality affects binding for duplex DNA but not for ssDNA. Taken together, these studies reveal different modes of binding for ss- and duplex DNA, with different driving forces, but in each case peptide structure contributes significantly to binding.;In another study, a beta-sheet peptide based on a WW domain sequence was redesigned for the molecular recognition of ssDNA. A previous report showed that (WKWK)2 binds ssDNA with low micromolar affinity but with little selectivity over dsDNA. This work extends those studies to a three-stranded beta-sheet designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows enhanced selectivity for ssDNA. The redesigned peptide no longer binds its native ligand, the polyproline helix. This indicates that the peptide has been redesigned for the function of binding ssDNA.;Structural studies indicate that this peptide consists of a structured beta-hairpin made of Strands 2&3 with a less structured strand 1, which provides affinity for ssDNA but does not improve the stability of the full peptide. Both function and stability are gained by incorporating a novel binding pocket into the peptide, and the redesigned peptide successfully mimics the OB-fold domain. Further mutations were made to design a mutant with increased structure, affinity, and selectivity for ssDNA. Knowledge gained from these binding and structural studies may lead to better designs of beta-sheet peptides designed to target nucleotides, damaged DNA, and ssDNA.