Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction

Abstract

We have determined the solution structure of the complex between an arginine-glycine-rich RGG peptide from the human fragile X mental retardation protein (FMRP) and an in vitro-selected guanine-rich (G-rich) sc1 RNA. The bound RNA forms a newly discovered G-quadruplex separated from the flanking duplex stem by a mixed junctional tetrad. The RGG peptide is positioned along the major groove of the RNA duplex, with the G-quadruplex forcing a sharp turn of R(10)GGGGR(15) at the duplex-quadruplex junction. Arg10 and Arg15 form cross-strand specificity-determining intermolecular hydrogen bonds with the major-groove edges of guanines of adjacent Watson-Crick G•C pairs. Filter-binding assays on RNA and peptide mutations identify and validate contributions of peptide-RNA intermolecular contacts and shape complementarity to molecular recognition. These findings on FMRP RGG domain recognition by a combination of G-quadruplex and surrounding RNA sequences have implications for the recognition of other genomic G-rich RNAs.

Figure 1

Sequence and NMR spectra of sc1 RNA and RGG peptide. (a) Sequence of 36-mer sc1 stem-loop RNA and 28-mer FMRP RGG peptide. (b,c) ¹H,¹⁵N HSQC spectra of RGG peptide in the (b) free and (c) sc1 RNA-bound states. Backbone amide resonance assignments are included in panel c. (d–f) Imino proton spectra (10.0 to 14.0 ppm) of (d) free sc1 RNA, (e) sc1 RNA bound to the RGG peptide, and (f) RGG peptide - sc1 RNA complex after 16 hr in D₂O. Spectra were recorded in 50 mM K-acetate, pH 6.8 at 25 °C.

Figure 2

RNA resonance assignments in the RGG peptide - sc1 RNA complex. (a) Example of imino proton assignment: the reference imino proton NMR spectrum of the complex is shown on the top and the corresponding filtered spectrum of the complex following site-specific incorporation of 2% ¹⁵N-labeled deoxyguanine at the G31 position in the RNA is shown at the bottom. (b) Guanine H8 protons were assigned by through-bond connectivities to the guanine imino protons via ¹³C5 by HMBC experiment.

Figure 3

Identification of G-tetrad alignments and assignment of through-bond correlations in the RGG peptide - sc1 RNA complex. (a) HNN-COSY contour plot showing through-bond connectivities between amino nitrogens and H8 protons around the G-tetrad. The labeling represents through-bond amino/H8 correlations between adjacent guanines within a G-tetrad plane. (b) HNN-COSY contour plot showing through-bond connectivities between amino protons and N7 nitrogens around the G-tetrad. The labeling represents through-bond amino/N7 correlations between adjacent guanines within a G-tetrad plane. (c) Hydrogen bonding alignment around the G-tetrad and data analysis supporting formation of three G-tetrads. (d) NOE connectivities between guanine imino and H8 protons around the G-tetrad. The labeling represents through-space imino/H8 correlations between adjacent guanines within a G-tetrad plane. (e) Identification of intermolecular ¹H-¹⁵N through-bond correlations in HNN-COSY spectra between guanine H8 protons of RNA and arginine guanidinium group nitrogens in the complex. Other correlations present in the spectra are intramolecular through-bond connectivities. Peak labeled a is between the H8 proton of G31 and Nε nitrogen of R10, while peak labeled b is between the H8 proton of G7 and the Nη nitrogen of R15. (f) Identification of intermolecular ¹H-¹H through bond correlations in HNN-COSY spectra between guanine H8 protons of RNA and arginine guanidinium group protons in the complex. Peak labeled a is between the H8 proton of G31 and NεH proton of R10, while peaks labeled b are between the H8 proton of G7 and the NηH amino protons of R15. Other correlations present in the spectra are intramolecular through-bond connectivities.

Figure 4

Solution structure of the RGG peptide - sc1 RNA complex and the architecture of the G-quadruplex and duplex-quadruplex junction. (a) Stereo views of 10 superpositioned refined structures of the RGG peptide - sc1 RNA complex. The bound peptide is colored in red, while the duplex, junction and quadruplex segments are colored in magenta, orange and cyan, respectively. The sugar-phosphate backbone of the RNA is in silver, with phosphorus atoms in yellow. Key arginine (R10 and R15) side chains are colored in green. (b) A representative refined structure of the RGG peptide - sc1 RNA complex with the same color coding as in panel a. (c) Location of a potential K⁺ binding site in the vicinity of the duplex-quadruplex junction following dynamics computations in a water bath containing K⁺ cations. Note that this K⁺ cation is anchored through interaction with oxygens from four phosphate groups.

Figure 5

Architecture of the G-quadruplex and duplex-quadruplex junction. (a) Schematic of the pairing alignments and strand connectivities at the duplex (magenta)-quadruplex (cyan) junction mediated by a mixed tetrad (orange). (b) Ribbon representation of schematic in panel a. (c and d). Base alignments around the junctional U8•A17•U28•G29 tetrad in the solution structure of the complex. 4 refined structures showed the pairing alignment in panel c, while 6 others showed the pairing alignment in panel d. (e) Schematic of pairing alignments and strand connectivities within the G-quadruplex. (f) Ribbon representation of schematic in panel e.

Figure 6

Details of intermolecular peptide-RNA interactions in the solution structure of the RGG peptide - sc1 RNA complex. (a) Positioning of the R10 to R15 segment of the RGG peptide (stick representation) within the major groove of the duplex segment of the sc1 RNA in the complex (surface representation). (b) A blow up emphasizing the intermolecular interaction shown in panel a. The peptide residues are labeled from R10 to R15. (c, d) Two views highlighting the intermolecular hydrogen bonding interactions between the guanidinium groups of R10 and R15 with the major groove edges of G31 and G7, respectively.

Figure 7

Assessment of the molecular determinants of the peptide and of the RNA for the FMRP RGG peptide - sc1 RNA interaction by filter binding assay. The affinity of interaction of the FMRP RGG box with 35-mer sc1 RNA (red) and mutations therein was determined by filter binding assay using the indicated concentrations of purified GST-tagged RGG box and ³²P end-labeled commercially synthesized RNA. (a) Mutation of Arg15 to Ala, Lys or Leu abolished sc1 RNA binding by the peptide. (b) Mutation of the glycines surrounding these crucial arginines. (WT RGG (red circles, K_d = 3.8 nM), Gly12Ala (open squares, K_d = ND), Gly13Ala (diamonds, K_d = ND), Gly14Ala (half closed squares, K_d = 49.5 nM), Gly16Ala (crossed squares, K_d = ND). (c) Mutation of nucleotides involved in the functional tetrads including U8C, A17U and U27A, U28A resulted in affinities too low to be measured. (d) The affinity of the RGG box for synthetic RNAs incorporating deaza-guanine either singly or in combination in positions G7 and G31 in sc1 RNA was measured to assess the importance of the N7 nitrogen of these guanines. Mutation of either N-7 (G7, open black squares, G31, diamonds) decreased binding by more than two orders of magnitude relative to the WT RNA (red circles) and the double mutation (half-closed black squares) decreased binding to an undetectable level.