d925f7fe1b9d6909effebe4925b67aa1 236604.pdf aaee1a5df4bc769384fe16dfa5e66086b622a626 236604.pdf 44b3ca0adb32e029f438b265f989f80d17de0c0735ef18e29b45ddcb36a23764 236604.pdf Title: Molecular Context-Dependent Effects Induced by Rett Syndrome-Associated Mutations in MeCP2 Subject: Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context. Keywords: Methyl-CpG-binding protein 2 (MeCP2); Rett syndrome; intrinsically disordered protein (IDP); protein stability; protein-DNA interaction; isothermal titration calorimetry (ITC) Author: David Ortega-Alarcon, Rafael Claveria-Gimeno, Sonia Vega, Olga C. Jorge-Torres, Manel Esteller, Olga Abian and Adrian Velazquez-Campoy Creator: LaTeX with hyperref package Producer: pdfTeX-1.40.18 CreationDate: Wed Nov 11 09:02:52 2020 CET ModDate: Wed Nov 25 09:03:11 2020 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3265951 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ZCRNCI+Pxsy Type 1 Builtin yes yes no 10 0 EIFDAO+VnURWPalladioL Type 1 Custom yes yes no 14 0 WOGFUG+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 NIASDU+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 WOZBHT+Rpxb Type 1 Builtin yes yes no 28 0 ZCYJJF+URWPalladioL-Roma Type 1 Custom yes yes no 32 0 SVVJCD+Rpxr Type 1 Builtin yes yes no 36 0 CUAJJQ+Pxbsy Type 1 Builtin yes yes no 40 0 IVVSWS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 50 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edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Plasmid Construction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Protein Expression and Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Double-Stranded DNA Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Circular Dichroism Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Fluorescence Spectroscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Isothermal Titration Calorimetry (ITC) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Children: Item: Title: Mutation R106W Induces Larger Structural Rearrangements Compared to R133C Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: RTT-Associated Mutations Alter Protein Stability and the dsDNA-Induced Stabilization Effect Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Stability Changes on MBD Induced by RTT-Associated Mutations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Stability Changes in RTT-Associated Mutant Proteins Induced by ds-DNA Binding Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: RTT-Associated Mutations Affect dsDNA Interaction Differently Depending on the MeCP2 Construction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Children: Item: Title: Interaction of MBD Mutants with ds-DNA Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Interaction of NTD-MBD-ID Mutants with ds-DNA Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Info: Title: Molecular Context-Dependent Effects Induced by Rett Syndrome-Associated Mutations in MeCP2 Author: David Ortega-Alarcon, Rafael Claveria-Gimeno, Sonia Vega, Olga C. Jorge-Torres, Manel Esteller, Olga Abian and Adrian Velazquez-Campoy Subject: Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context. 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