Crystal structure reveals specific recognition of a G-quadruplex RNA by a β-turn in the RGG motif of FMRP

Significance The arginine-glycine-rich (RGG) box is an abundant RNA-binding motif present in many proteins. The RGG motif of the Fragile Mental Retardation Protein (FMRP) specifically binds guanine-quadruplex-containing RNA. Using X-ray crystallography, we uncovered molecular principles that account for specificity and affinity of the interactions between the RGG motif and guanine-quadruplex-containing RNA. The structure-based biochemical assays revealed that specific recognition of the RNA extends to the duplex region, thus identifying a broad network of important RNA–protein interactions and suggesting a common RNA binding principle for other RGG motif-containing proteins. Fragile X Mental Retardation Protein (FMRP) is a regulatory RNA binding protein that plays a central role in the development of several human disorders including Fragile X Syndrome (FXS) and autism. FMRP uses an arginine-glycine-rich (RGG) motif for specific interactions with guanine (G)-quadruplexes, mRNA elements implicated in the disease-associated regulation of specific mRNAs. Here we report the 2.8-Å crystal structure of the complex between the human FMRP RGG peptide bound to the in vitro selected G-rich RNA. In this model system, the RNA adopts an intramolecular K+-stabilized G-quadruplex structure composed of three G-quartets and a mixed tetrad connected to an RNA duplex. The RGG peptide specifically binds to the duplex–quadruplex junction, the mixed tetrad, and the duplex region of the RNA through shape complementarity, cation–π interactions, and multiple hydrogen bonds. Many of these interactions critically depend on a type I β-turn, a secondary structure element whose formation was not previously recognized in the RGG motif of FMRP. RNA mutagenesis and footprinting experiments indicate that interactions of the peptide with the duplex–quadruplex junction and the duplex of RNA are equally important for affinity and specificity of the RGG–RNA complex formation. These results suggest that specific binding of cellular RNAs by FMRP may involve hydrogen bonding with RNA duplexes and that RNA duplex recognition can be a characteristic RNA binding feature for RGG motifs in other proteins.