STRUCTURAL BASIS OF NUCLEIC ACID RECOGNITION AND PHASE SEPARATION BY THE PLANT GLYCINE-RICH PROTEIN AtGRP2

AtGRP2 (Arabidopsis thaliana glycine-rich protein 2) is a glycine-rich, RNA-binding protein that plays key roles in embryogenesis, flowering, and abiotic stress response in Arabidopsis thaliana. AtGRP2 consists of an N-terminal cold shock domain (CSD) and two C-terminal retroviral-like CCHC-type zinc knuckles interspersed with glycine-rich regions. Despite the wealth of information on AtGRP2 biological roles, the molecular mechanisms underlying its functions are largely unknown. Here, we investigated the structure, dynamics, and nucleic acid binding properties of AtGRP2-CSD. In addition, we characterized the liquid-liquid phase separation propensity of full-length AtGRP2. AtGRP2-CSD_1-79 undergoes a folded-unfolded equilibrium that is shifted toward the folded state upon DNA binding. Addition of eleven residues at the C-terminus stabilized the folded conformation. AtGRP2-CSD_1-90 revealed a b-barrel structure composed of five b-strands (b1 to b5) and a well-defined C-terminal extension that stabilizes the CSD fold. AtGRP2-CSD_1-90 uses a preformed platform of solvent-exposed hydrophobic residues, located in strands b2, b3, and the b3-b4 loop, to preferentially interact with T-rich DNA. In addition, T7 binding decreased the b3-b4 loop flexibility, while increasing residues Lys22 and Arg49 conformational exchange, suggesting that they may compose an encounter protein:DNA complex. Full-length AtGRP2 forms spherical, micron-sized droplets in conditions of molecular crowding. AtGRP2 condensates do not fuse, do not recover fluorescence in FRAP experiments, and form sticky structures over time, suggesting gel-like material properties. Hydrophobic instead of electrostatic forces are the main drivers of AtGRP2 condensation, most likely p-p interactions between Tyr pairs in the C-terminal glycine-rich intrinsically disordered tail. RNA, either a total extract from A. thaliana or a 30-nucleotide Lin28-specific sequence, maintains AtGRP2 in the diffuse state. Altogether, these results shed light on the mechanism of nucleic acid recognition and phase separation employed by AtGRP2, creating a framework for the development of biotechnological strategies to increase plant crop resistance to multiple abiotic stresses.