| Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability in human. Absence of fragile X mental retardation protein (FMRP) is the cause of FXS. FMRP is involved in different steps of RNA metabolism through selectively binding to RNA. Of particular interest is FMRP’s role in translation regulation. Although three models for how FMRP inhibits translation have been proposed, it is also reported that FMRP acts in translational activation. Absence of FMRP causes translation, transport or stability of its targeted mRNA to be unregulated, leading to a cascade effect on many neurological pathways and finally FXS. At the molecular level, FMRP uses two central KH domains and a C-terminal Arginine-Glycine-Glycine (RGG) box to recognize RNA, such as G quadruplex RNA. However, there are essential questions in N terminal domain concerning functions and structure of FMRP. For instance, a region (135-217 residues) is involved in FMRP’s interactions with many proteins or nuclear acids, but its structure is still undetermined. Moreover, the oligomerization property of FMRP N terminal domain is related to FMRP cellular functions, but mechanisms behind it remain unknown. Recently, FMRP N terminal domain is declared to bind chromatin and this interaction is critical for FMRP function in DNA damage response. However, whether and how FMRP recognizes a particular histone post-translational-modification are both unclear.Through screening of truncated variants containing different length of amino acid residues or number of domains, the human FMRP N terminal protein was obtained. The FMR1-209 high-quality crystals were acquired by using one material named precipitant-immobilized Molecular Imprinted Polymers (piMIPs) as an additive. The 3D structure of FMRP1-209 crystal was determined by molecular replacement (FMRP-NT). FMRP-NT contains two inter-domain long loops and three domains, adopting an approximately linear architecture model. We discovered that the 127-200 residues of FMRP-NT exists as an independent fold. Bioinformatics analysis and structural comparison revealed that it is a new subtype of KH domain and we named it KHO. Because the highly conserved RNA-recognizing "GXXG" motif and two isoleucines in typical KH domains are all absent in it, we speculated that KHO owns different functions from other KH domains. In an asymmetric unit of FMRP-NT crystal, we found two intermolecular disulfide bonds (Cys99-Cys99), by which two hydrophobic interfaces are mediated. In this study, we determined the crystal structure of FMRP Tudorl-Tudor2 domains in the N terminus for the first time. By comparing them with the determined NMR Tudorl and Tudor2 structure respectively, we verified that piMIPs didn’t alter these two folds, and the application of piMIPs in FMRP-NT crystal optimization proved to be effective. Analysis of the overall structure of FMRP-NT emphasized the importance of some residues in Tudorl domain in maintaining stability through domain-domain and domain-loop interactions. Besides, X-ray Small Angle Scattering, analytical ultracentrifugation and gel filtration chromatography experiments of FMRP-NT wild type and structure-guided mutants showed that FMRP-NT dimers have several kinds of conformations in solution, and the key residue Ile 106 for FMRP-NT dimerization is discovered. Moreover, in our preliminary study of FMRP’s role in histone post-translational-modification recognition, we found obvious binding between FMRP N terminal domain and a modified histone peptide named E20. Microscale thermophoresis experiments and isothermal titration calorimetry experiments both revealed that the C terminal non-modified 10 residues is required for the interaction of FMRP and E20, although the binding affinity is not high.In this study, we discover KHO domain in FMRP and reveal FMRP N terminal domain forms several kinds of dimers in solution and binds a modified histone peptide. These findings provide new evidence to elucidate the functions of FMRP in cell. |
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