Structural basis of nucleic acid recognition by the N-terminal cold shock domain of the plant glycine-rich protein AtGRP2

AtGRP2 is a glycine-rich, RNA-binding protein that plays pivotal roles in abiotic stress response and fl owering time regulation in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal CCHC-type zinc knuckles interspersed with glycine-rich regions. Here, we investigated the structure, dynamics, and nucleic acid-binding properties of AtGRP2-CSD. The 2D [1H,15N] heteronuclear single quantum coherence spectrum of AtGRP2-CSD1-79 revealed the presence of a partially folded intermediate in equilibrium with the folded state. The addition of 11 residues at the C terminus stabilized the folded conformation. The three-dimensional structure of AtGRP2-CSD1-90 unveiled a (3-barrel composed of fi ve antiparallel (3-strands and a 3 10 helical turn, along with an ordered C-terminal extension, a conserved feature in eukaryotic CSDs. Direct contacts between the C-terminal extension and the (33-(34 loop further stabilized the CSD fold. AtGRP2-CSD1-90 exhibited nucleic acid binding via solvent-exposed residues on strands (3 2 and (3 3, as well as the (33-(34 loop, with higher affinity for DNA over RNA, particularly favoring pyrimidine-rich sequences. Furthermore, DNA binding induced rigidity in the (33-(34 loop, evidenced by 15 N-{1H} NOE values. Mutation of residues W17, F26, and F37, in the central (3-sheet, completely abolished DNA binding, highlighting the significance of 7L-stacking interactions in the binding mechanism. These results shed light on the mechanism of nucleic acid recognition employed by AtGRP2, creating a framework for the development of biotechnological strategies aimed at enhancing plant resistance to abiotic stresses.