Identification\nof G‑Quadruplex-Binding Protein\nfrom the Exploration of RGG Motif/G-Quadruplex Interactions

Abstract

The\narginine/glycine-rich region termed the RGG domain is usually\nfound in G-quadruplex (G4)-binding proteins and is important in G4–protein\ninteractions. Studies on the binding mechanism of RGG domains found\nthat small segments (RGG motif) inside the domain contribute greatly\nto the G4 binding affinity. However, unlike the entire RGG domains\nthat have been broadly explored, the role of the RGG motif remains\nobscure, with very limited study. Herein, to clarify the role of the\nRGG motif in G4–protein interactions, we systematically investigated\nthe binding affinity and mode between RGG-motif peptides and G4s.\nThe internal arrangement of RGG repeats and gap amino acids played\na more crucial role in the G4-binding mechanism than a critical number\nof RGG repeats. Arginines and phenylalanines at the exact position\nof the RGG motif might enable additional hydrogen bonding and π-stacking\ninteraction with nucleobases and strengthen the binding of G4. Impressively,\nproceeding from a G4-binding RGG peptide, <b>12</b>, discovered\nabove, we identified the cold-inducible RNA-binding protein (CIRBP)\nas a new G4 DNA-binding protein both in vitro and in cells. In addition,\nwe found that the key amino acids for G4 binding in peptide <b>12</b> and CIRBP were highly similar, and peptide <b>12</b> clearly played a key role in the G4 binding of CIRBP. This report\nis the first in which a G4-binding protein was identified from exploration\nof the G4-binding RGG motif. Our findings suggest a novel strategy\nfor discovering new G4-binding proteins by exploring key peptide segments.