Structural characterization and DNA binding properties of the RRM domain of the plant glycine-rich protein AtGRP7

AtGRP7 (Arabidopsis thaliana glycine-rich protein 7) is a glycine-rich, RNA-binding
protein that plays a crucial role in plant growth, development, and abiotic stress
response. AtGRP7 consists of an N-terminal RNA recognition motif (RRM) followed by an intrinsically disordered, glycine-rich region. Despite AtGRP7 significance in cold adaptation and flowering time regulation, the biochemical mechanisms underlying its function are largely unknown. Here, we employed biophysical techniques to investigate the structure, dynamics, and DNA binding properties of AtGRP7-RRM. The RMM domain of AtGRP7 (residues 1-90) was cloned into RP1B and expressed in Escherichia coli BL21 DE3 as a His 6 -fusion protein. Following purification by nickel-affinity and size exclusion chromatography, AtGRP7-RRM was characterized using circular dichroism, NMR and fluorescence spectroscopy. Circular dichroism revealed features consistent of a typical RRM fold, comprising a mixture of α-helices and β-sheets. AtGRP7-RRM displayed a melting temperature of 38 o C, indicating relatively low stability. Using multidimensional, triple resonance NMR, we unambiguously assigned 90% of the backbone resonances. Notably, some NH resonances were missing for residues Met1, Ala2, Ser3, Gly4, Trp17, Arg43, Gln85, Ser86, Arg87, Gly88, Ser89, Gly90, likely due to conformational dynamics or solvent exchange. Upon titration of a 7-nucleotide DNA ligand, previously identified as a specific binding site, we observed shifts in amide resonances. Analysis of the binding interface revealed that surface-exposed residues in the 2-3 loop and strands 1 and 5 are involved in DNA interaction. The fluorescence emission spectrum of AtGRP7-RRM exhibited a maximum at ~349 nm, indicating solvent exposure of Trp17. Increasing concentrations of DNA resulted in fluorescence quenching, and AtGRP7-RRM interaction with DNA occurred with an apparent K D of 17.9 ± 4.2 µM. 15 N relaxation parameters indicated fast backbone motions in the 2-3 loop, suggesting that loop flexibility is correlated to DNA binding. This is an important first step toward the structure determination of AtGRP7- RRM, which will shed light into its DNA recognition mechanism.

Financial Support: FAPERJ, CNPq, CAPES