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• W1970149098 abstract "Most cases of Rett syndrome (RTT) are caused by mutations in the methylated DNA-binding protein, MeCP2. Here, we have shown that frequent RTT-causing missense mutations (R106W, R133C, F155S, T158M) located in the methylated DNA-binding domain (MBD) of MeCP2 have profound and diverse effects on its structure, stability, and DNA-binding properties. Fluorescence spectroscopy, which reports on the single tryptophan in the MBD, indicated that this residue is strongly protected from the aqueous environment in the wild type but is more exposed in the R133C and F155S mutations. In the mutant proteins R133C, F155S, and T158M, the thermal stability of the domain was strongly reduced. Thermal stability of the wild-type protein was increased in the presence of unmethylated DNA and was further enhanced by DNA methylation. DNA-induced thermal stability was also seen, but to a lesser extent, in each of the mutant proteins. Circular dichroism (CD) of the MBD revealed differences in the secondary structure of the four mutants. Upon binding to methylated DNA, the wild type showed a subtle but reproducible increase in α-helical structure, whereas the F155S and R106W did not acquire secondary structure with DNA. Each of the mutant proteins studied is unique in terms of the properties of the MBD and the structural changes induced by DNA binding. For each mutation, we examined the extent to which the magnitude of these differences correlated with the severity of RTT patient symptoms. Most cases of Rett syndrome (RTT) are caused by mutations in the methylated DNA-binding protein, MeCP2. Here, we have shown that frequent RTT-causing missense mutations (R106W, R133C, F155S, T158M) located in the methylated DNA-binding domain (MBD) of MeCP2 have profound and diverse effects on its structure, stability, and DNA-binding properties. Fluorescence spectroscopy, which reports on the single tryptophan in the MBD, indicated that this residue is strongly protected from the aqueous environment in the wild type but is more exposed in the R133C and F155S mutations. In the mutant proteins R133C, F155S, and T158M, the thermal stability of the domain was strongly reduced. Thermal stability of the wild-type protein was increased in the presence of unmethylated DNA and was further enhanced by DNA methylation. DNA-induced thermal stability was also seen, but to a lesser extent, in each of the mutant proteins. Circular dichroism (CD) of the MBD revealed differences in the secondary structure of the four mutants. Upon binding to methylated DNA, the wild type showed a subtle but reproducible increase in α-helical structure, whereas the F155S and R106W did not acquire secondary structure with DNA. Each of the mutant proteins studied is unique in terms of the properties of the MBD and the structural changes induced by DNA binding. For each mutation, we examined the extent to which the magnitude of these differences correlated with the severity of RTT patient symptoms. A key epigenetic signal in vertebrates is the symmetrical methylation of CpG dinucleotides, which may be passed on to subsequent generations by the action of hemi-methylases on newly replicated DNA (reviewed in Ref. 1Klose R.J. Bird A.P. Trends Biochem. Sci. 2006; 31: 91-97Abstract Full Text Full Text PDF Scopus (1909) Google Scholar). Screening for proteins that bind preferentially to methylated CpGs has revealed a family of methylated DNA-binding proteins, the founding member of which is the conserved and highly basic 52-kDa methylated DNA-binding protein 2, MeCP2 2The abbreviations used are: MeCP2, methylated DNA-binding protein 2; RTT, Rett syndrome; MBD, methylated DNA-binding domain; EMSA, electrophoretic mobility shift assay; WT, wild type; BDNF, brain-derived neurotrophic factor. 2The abbreviations used are: MeCP2, methylated DNA-binding protein 2; RTT, Rett syndrome; MBD, methylated DNA-binding domain; EMSA, electrophoretic mobility shift assay; WT, wild type; BDNF, brain-derived neurotrophic factor. (reviewed in Ref. 2Wade P.A. BioEssays. 2001; 23: 1131-1137Crossref PubMed Scopus (289) Google Scholar). The portion of MeCP2 responsible for binding methylated DNA is known as the MBD (methylated DNA-binding domain), which extends from residues ∼75 to ∼164 (3Nan X. Meehan R.R. Bird A. Nucleic Acids Res. 1993; 21: 4886-4892Crossref PubMed Scopus (490) Google Scholar). NMR and x-ray studies (4Wakefield R.I.D. Smith B.O. Nan X. Free A. Soteriou A. Uhrin D. Bird A.P. Barlow P.N. J. Mol. Biol. 1999; 291: 1055-1065Crossref PubMed Scopus (167) Google Scholar, 5Ho K.L. McNae I.W. Schmiedeberg L. Klose R.J. Bird A.P. Walkinshaw M.W. Mol. Cell. 2008; 29: 525-531Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) have shown the MBD to be ∼60% structured, with segments of α-helix, β-strand, and β-turn forming a wedge-shaped structure (Fig. 1b). In contrast, the N- and C-terminal portions of MeCP2 are predicted to be largely unstructured (6Adams V.H. McBryant S.J. Wade P.A. Woodcock C.L. Hansen J.C. J. Biol. Chem. 2007; 282: 15057-15064Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Signals encoded in methylated CpGs frequently lead to transcriptional repression, which appears to be a prominent consequence of MeCP2 binding (7Nan X. Campoy F.J. Bird A. Cell. 1997; 88: 471-481Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar). One model of the mechanism that leads from MeCP2 binding to transcriptional repression involves the recruitment of Sin 3A and histone deacetylase followed by local histone modification (8Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2237) Google Scholar, 9Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2770) Google Scholar). However, recent evidence suggests that MeCP2 locations in chromatin are not confined to sites of methylated DNA and that MeCP2 occupancy does not necessarily lead to transcriptional repression (10Yasui D.H. Peddada S. Bieda M.C. Vallero R.O. Hogart A. Nagarajan R.P. Thatcher K.N. Farnham P.J. Lasalle J.M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 19416-19421Crossref PubMed Scopus (320) Google Scholar). Moreover, it is now clear that MeCP2 has a wide range of potential functions (reviewed in Refs. 11LaSalle J.M. Curr. Top. Dev. Biol. 2004; 59: 61-86Crossref PubMed Scopus (13) Google Scholar and 12Bienvenu T. Chelly J. Nat. Rev. Genet. 2006; 7: 415-426Crossref PubMed Scopus (230) Google Scholar), including an involvement in RNA processing (13Young J.I. Hong E.P. Castle J.C. Crespo-Barreto J. Bowman A.B. Rose M.F. Kang D. Richman R. Johnson J.M. Berget S. Zoghbi H.Y. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17551-17558Crossref PubMed Scopus (359) Google Scholar) and a regulatory role in several human cancers (14Bernard D. Gil J. Dumont P. Rizzo S. Monte D. Quatannens B. Hudson D. Visakorpi T. Fuks F. de Launoit Y. Oncogene. 2006; 25: 1358-1366Crossref PubMed Scopus (45) Google Scholar, 15Muller H.M. Fiegl H. Goebel G. Hubalek M.M. Widschwendter A. Muller-Holzner E. Marth C. Widschwendter M. Br. J. Cancer. 2003; 89: 1934-1939Crossref PubMed Scopus (58) Google Scholar, 16Sharma D. Blum J. Yang X. Beaulieu N. Macleod A.R. Davidson N.E. Mol. Endocrinol. 2005; 19: 1714-1751Google Scholar). MeCP2 misregulation has also been found associated with autism spectrum disorders (17Nagarajan R.P. Hogart A.R. Gywe Y. Martin M.R. LaSalle J.M. Epigenetics. 2006; 1: e1-e11Crossref PubMed Scopus (271) Google Scholar). Our previous work on the in vitro interactions between MeCP2 and chromatin suggests that it is a potent inducer of compaction and thus may contribute to transcriptional repression via conformational changes to chromatin (18Georgel P.T. Horowitz-Scherer R.A. Adkins N. Woodcock C.L. Wade P.A. Hansen J.C. J. Biol. Chem. 2003; 278: 32181-32188Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 19Nikitina T.N. Shi X. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. Mol. Cell. Biol. 2007; 27: 864-877Crossref PubMed Scopus (143) Google Scholar, 20Nikitina T.N. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. J. Biol. Chem. 2007; 282: 28237-28245Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 21Chadwick L.H. Wade P.A. Curr. Opin. Genet. Dev. 2007; 17: 1-5Crossref Scopus (35) Google Scholar). An important landmark in understanding the in vivo role of MeCP2 was the finding that most cases of Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder in humans, are attributable to sporadic mutations in MeCP2 (22Amir R.E. den Veyber I.B. Wan M. Tran C.Q. Francke U. Zoghbi H.Y. Nat. Genet. 1999; 23: 185-188Crossref PubMed Scopus (3799) Google Scholar). Females hemizygous for mutated MeCP2 develop normally for 6-9 months and then show progressive neurological dysfunctions that appear to be associated with a loss of dendritic complexity and reduction in brain size (reviewed in Ref. 23Shabazian M.D. Zoghbi H. Am. J. Hum. Genet. 2002; 71: 1259-1272Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). There is a wide variation in the range and severity of RTT symptoms, which appears to be related, at least in part, to the specific mutation present and to local X inactivation skewing. MeCP2 mutations in males produce a range of symptoms ranging from mild mental retardation to severe neonatal encephalopathy (reviewed in Ref. 24Villard L. J. Med. Genet. 2007; 44: 417-423Crossref PubMed Scopus (125) Google Scholar). Further, deletion of MeCP2 in mice gives rise to RTT-like symptoms, which can be reversed upon expression of an introduced copy of the MeCP2 gene (25Guy J. Gan J. Selfridge J. Cobb S. Bird A. Science. 2007; 315: 1143-1147Crossref PubMed Scopus (876) Google Scholar). The clear association between MeCP2 and neurological diseases provides an opportunity to understand the mechanism by which MeCP2 selectively interacts with the genome and transmits methylated DNA signals. The majority of RTT cases result either from C-terminal truncations of varying length (∼40% of cases) or missense mutations within the MBD (∼45% of cases). Common MBD mutations include T158M (∼10% of patients), R106W (∼4%), and R133C (∼4%) (26Philippe, C., Villard, L., DeRoux, N., Raynaud, M., Bonnefond, J. P., Pasquier, L., Lesca, G., Mancini, J., Jonveaux, P., Moncla, A., Chelly, J., and Bienvenu, T. (2006) Eur. J. Med. Genet. 49, 9-18Google Scholar, 27Percy A.K. J. Child Neurol. 2007; 20: 1-7Google Scholar) (www.mecp2.org.uk/). The x-ray structure of an MeCP2 MBD-DNA complex reveals that Arg-133 is involved in the DNA interaction surface (5Ho K.L. McNae I.W. Schmiedeberg L. Klose R.J. Bird A.P. Walkinshaw M.W. Mol. Cell. 2008; 29: 525-531Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), a result consistent with an earlier NMR study using the homologous domain of the methyl-binding MBD1 protein (28Ohki I. Shimotake N. Fujita N. Jee J.-G. Ikegami T. Nakao M. Shirakawa M. Cell. 2001; 105: 487-497Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Other frequent RTT-causing MBD mutations are not directly associated with the DNA interaction interface, and the causes of their loss of function appear to involve changes in inter-residue interactions (5Ho K.L. McNae I.W. Schmiedeberg L. Klose R.J. Bird A.P. Walkinshaw M.W. Mol. Cell. 2008; 29: 525-531Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Our recent studies of the interactions between MeCP2 and chromatin (19Nikitina T.N. Shi X. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. Mol. Cell. Biol. 2007; 27: 864-877Crossref PubMed Scopus (143) Google Scholar, 20Nikitina T.N. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. J. Biol. Chem. 2007; 282: 28237-28245Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) revealed multiple DNA binding sites that extend beyond the MBD and involve both methylated and unmethylated DNA. We have also shown that different RTT-causing mutations have a surprisingly diverse impact on DNA and chromatin binding and compaction, suggesting that a comparative study of the effects of the different mutants on MeCP2 structure and substrate binding would be highly informative. We have therefore embarked on a detailed examination of the contributions of the different domains of wild type and mutant and the impact of DNA binding on the conformation of the protein. We chose first to study the MBD because the preponderance of missense mutations found in RTT patients occurs there. We selected four RTT-causing missense MBD mutations (Fig. 1) for detailed study, including the three that occur most frequently. The impact of these mutations on the secondary and tertiary structure of the MBD and full-length protein were compared using circular dichroism (CD) and fluorescence spectroscopy. We show that each of these MBD mutations has unique structural consequences relevant to their loss of function. Within the intrinsically unstructured MeCP2 protein, protease-protected domains can be identified beyond the structured MBD (6Adams V.H. McBryant S.J. Wade P.A. Woodcock C.L. Hansen J.C. J. Biol. Chem. 2007; 282: 15057-15064Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and an important finding of the present study is evidence of coupling between domains. Plasmid Constructs for Recombinant MeCP2 and Mutants—Plasmids containing genes for wild-type human MeCP2 and the mutants R133C and F155S fused to a chitin-binding domain were kindly supplied by Paul Wade and Timur Yusufzai. The mutants R106W and T158M were generated by site-directed mutagenesis using the following primers and the QuikChange® II XL site-directed mutagenesis kit: R106W forward, 5′-CCACCCTGCCTGAAGGCTGGACATGGAAGCTTAAGCAAAGG-3′; R106W reverse, 5′-CCTTTGCTTAAGCTTCCATGTCCAGCCTTCAGGCAGGGTGG-3′; T158M forward, 5′-GGACCCTAATGATTTTGACTTCATGGTAACTGGGAGAGGGAGCCCC-3′; T158M reverse, 5′-GGGGCTCCCTCTCCCAGTTACCATGAAGTCAAAATCATTAGGGTCC-3′. For constructing the MBD (residues 75-164), amplicons with 5′-NdeI and 3′-EcoRI linker and additional hexanucleotide overhangs at each end were engineered using the following primer pair: MBD forward primer, 5′-CAATGACATATGGAAGCTTCTGCCTCCCCCAAACAGC-3′; MBD reverse primer, 5′-GTTAGAGAATTCGCTCCCTCTCCCAGTTACCGTGAAG-3′. The MBD mutants (R106W/MBD, T158M/MBD, F155S/MBD, R133C/MBD) were engineered using the full-length MeCP2 variants as templates and the MBD forward and reverse primer couples in amplification reaction. For constructing MeCP2 fragments 1-164 (MBD plus N-terminal flanking 74 amino acids) and MeCP2-(1-294), the forward primer 5′-CCGGTTTAAACCGGGGATCTCGATCC-3′ was used in combination with the reverse primers 5′-GTTAGAGAATTCGCTCCCTCTCCCAGTTACCGTGAAG-3′ and 5′-GACCGTGAATTCTCGGATAGAAGACTCCTTCACG-3′, respectively. This produced amplicons with a 5′ ∼100-bp extension into the pTYB1 vector bearing an NdeI site overlapping with the start codon of the protein. Fragment-(75-209) (MBD plus C-terminal flanking 45 amino acids) was constructed using the MBD forward primer and the following reverse primer: 5′-GTTAGAGAATTCCACCTGCACACCCTCTGACGTGGC-3′. The restriction fragments generated by double digestion of the amplicons with NdeI + EcoRI, were then cloned into pTYB1 (New England Biolabs) vector by standard ligation procedures. Protein Purification—Full-length MeCP2 (WT and the mutants R133C, F155S, T158M AND R106W), the fragments MeCP2-(1-164), MeCP2-(75-209), and MeCP2-(1-294), and MBD (WT and mutants) were purified using the IMPACT system (New England Biolabs) as described (19Nikitina T.N. Shi X. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. Mol. Cell. Biol. 2007; 27: 864-877Crossref PubMed Scopus (143) Google Scholar). For MBD mutants, protein was applied to heparin HP columns in 100 mm (instead of 250 mm) NaCl and eluted using salt steps from 0.1 to 1.0 m NaCl at increments of 0.1 m. DNA Preparation—Complementary strands of high pressure liquid chromatography-purified 45-bp segments of BDNF promoter IV of the mouse brain-derived neurotrophic factor (BDNF) gene, with and without methylation of the single cytosine, were obtained from Integrated DNA Technologies in an equimolar mix. Complementary Pair1 (5′-GCCATGCCCTGGAACGGAACTCTCCTAATAAAAGATGTATCATTT-3′/5′-AAATGATACATCTTTTATTAGGAGAGTTCCGTTCCAGGGCATGGC-3′) and Pair2 (5′-GCCATGCCCTGGAA(5′-Me)CGGAACTCTCCTAATAAAAGATGTATCATTT-3′/5′-AAATGATACATCTTTTATTAGGAGAGTTC(5′-Me)CGTTCCAGGGCATGGC-3′) were annealed using a modified touchdown protocol (Integrated DNA Technologies) on a PTC-100 thermal cycler (denaturation step at 95 °C for 2 min followed by incubation at 60 °C for 10 min and finally cooling to 4 °C at the rate of 1 °C every 15 s) to generate the unmethylated and methylated substrates. 601-12 DNA was prepared and methylated as described (20Nikitina T.N. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. J. Biol. Chem. 2007; 282: 28237-28245Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Fluorescence Spectroscopy—Fluorescence emission spectra were obtained using a PTI QM1 spectrofluorimeter over a 95 nm window from 305 to 400 nm using 2 nm emission and excitation slits with an integration time of 0.3 s. Three independent scans were averaged for each condition. For thermal unfolding, sample temperature was controlled using a Peltier unit, and samples were held at each temperature for 5 min before collection of data. Spectra were collected at 2.5 °C intervals between 5 or 10 °C and 85 °C The midpoints of the melting transitions (Tm) were obtained from least square fits of plots of relative fluorescence intensity at λmax versus temperature and from the peaks of first derivative plots. Both approaches yielded very similar Tm values. Relative fluorescence intensity was calculated by setting the intensity at the starting temperature to 100% and the ending temperature to 0% and interpolating values for intermediate temperatures. Experiments using protein + DNA were done in duplicate and, with protein only, in triplicate. Experiments with MeCP2-DNA complexes were carried out with protein:DNA molar ratios of 1:1 and 1:2 with protein concentrations between 2.5 and 5.0 μm. Both input ratios gave essentially identical results. Circular Dichroism—Proteins were first dialyzed extensively in 10 mm phosphate buffer, pH 7.6, containing 100 mm NaF. CD measurements were carried out at 22 °C with a J715CD spectropolarimeter (Jasco Inc.) at a bandwidth of 1 nm and spectral resolution of 0.5 nm using a 0.1-cm path length stoppered quartz cuvette (Sterna Inc.). The scanning rate was 20 nm/s in continuous scanning mode with a response time of 4 s. Five spectra were collected per protein, averaged, and buffer-subtracted. The mean residue ellipticity [θ] (degrees cm2 dmol-1 residue-1) was obtained by normalization of the raw data using [θ]=θobs×M/10·/·C(Eq. 1) where [θ] is the mean residue ellipticity (degrees cm2 dmol-1 residue-1), θobs is the ellipticity measured in degrees, M is the protein mean residue molecular weight, l is the optical path length of the cuvette in cm, and C is the concentration of the protein in mg/ml (29Del Vecchio P. Graziano G. Granata V. Barone L.M. Rossi M. Manco G. Biochem. J. 2002; 367: 857-863Crossref PubMed Google Scholar). In some cases, the proportions of the various types of secondary structure were estimated using CONTINLL supported by DICHROWEB, an online server for protein secondary structure analyses (30Whitmore L. Wallace B.A. Nucleic Acids Res. 2004; 32: W668-W673Crossref PubMed Scopus (1986) Google Scholar, 31Lobley A. Whitmore L. Wallace B.A. Bioinformatics (Oxf.). 2002; 18: 211-212Crossref PubMed Scopus (645) Google Scholar). For estimations using LINCOMB, data were fitted to the Greenfield-Fasman standard curve set of 17 proteins using constrained least squares minimization as described (32Perczel A. Park K. Fasman G.D. Anal. Biochem. 1992; 203: 83-91Crossref PubMed Scopus (421) Google Scholar). For estimations using CONTINLL (33Provencher S.W. Glockner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1878) Google Scholar, 34van Stokkum I.H. Spoelder H.J. Bloemendal M. van Grondelle R. Groen P.C. Anal. Biochem. 1990; 191: 110-118Crossref PubMed Scopus (437) Google Scholar), reference set 7 on Dichroweb, optimized for the wavelength range of 190 to 240 and containing 32 proteins from SELCON3, six from Ref. 33Provencher S.W. Glockner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1878) Google Scholar, five from Ref. 35Pancoska P. Bitto E. Janota V. Urbanova M. Gupta V.P. Keiderling T.A. Protein Sci. 1995; 4: 1384-1401Crossref PubMed Scopus (83) Google Scholar, and five denatured, was used. The estimated composition of the MBD calculated using LIN-COMB, CONTINLL was also compared with the known NMR-derived secondary structure of the MBD (Protein Data Bank code 1QK9 (4Wakefield R.I.D. Smith B.O. Nan X. Free A. Soteriou A. Uhrin D. Bird A.P. Barlow P.N. J. Mol. Biol. 1999; 291: 1055-1065Crossref PubMed Scopus (167) Google Scholar)) using STRIDE (36Frishman D. Argos P. Proteins. 1995; 23: 566-579Crossref PubMed Scopus (2037) Google Scholar). Electrophoretic Mobility Shift Assay (EMSA)—To examine DNA binding efficiency and methylation specificity, wild-type and mutant MBD protein was mixed with 200 ng of methylated or unmethylated 601-12 DNA (20Nikitina T.N. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. J. Biol. Chem. 2007; 282: 28237-28245Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) in the presence of 400 ng of unmethylated 208-1 competitor DNA (19Nikitina T.N. Shi X. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. Mol. Cell. Biol. 2007; 27: 864-877Crossref PubMed Scopus (143) Google Scholar) in binding buffer (100 mm NaCl, 10 mm Tris, 0.025% Nonidet P-40, 0.25 mm EDTA, pH 7.4) at room temperature for 30 min. Electrophoresis was performed on prechilled 1% agarose type IV gels, which were run at 85 V for 4 h at 4 °C in TAE (40 mm Tris, 24 mm acetic acid, 0.5 mm EDTA, pH 8.3) buffer (19Nikitina T.N. Shi X. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. Mol. Cell. Biol. 2007; 27: 864-877Crossref PubMed Scopus (143) Google Scholar). Molecular Modeling—Models of human MeCP2 MBD residues 77-165 were built and visualized with Insight II and Biopolymer software (Accelrys, San Diego, CA) and UCSF Chimera (University of California, San Francisco). The representative structure from the most highly populated ensemble cluster (37Kelley L.A. Gardner S.P. Sutcliffe M.J. Protein Eng. 1996; 9: 1063-1065Crossref PubMed Scopus (417) Google Scholar) of the NMR suite (4Wakefield R.I.D. Smith B.O. Nan X. Free A. Soteriou A. Uhrin D. Bird A.P. Barlow P.N. J. Mol. Biol. 1999; 291: 1055-1065Crossref PubMed Scopus (167) Google Scholar) (Protein Data Bank code 1QK9) was selected for this purpose. Amino acid substitutions made at the sites of the selected RTT mutations (R106W, R133C, F155S and T158M) were generated using the Richardson rotamer library (38Lovell S.C. Word J.M. Richardson J.S. Richardson D.C. Proteins. 2000; 40: 389-408Crossref PubMed Scopus (894) Google Scholar) and energy-minimized with MMTK (Molecular Modelling Toolkit) (39Hinsen K. J. Comput. Chem. 2000; 21: 79-85Crossref Scopus (270) Google Scholar) with the AMBER ff99 force fields (40Case D.A. Cheatham III, T.E. Darden T. Gohlke H. Luo R. Merz K.M. Onufriev A. Simmerling C. Wang B. Woods R. J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6519) Google Scholar). Solvent-accessible surface areas were calculated with GETAREA (41Fraczkiewicz R. Braun W. J. Comp. Chem. 1998; 19: 319-333Crossref Scopus (869) Google Scholar). RTT-causing Mutations Alter the Structure, Stability, and DNA-binding Properties of MeCP2—Wild-type MeCP2 has a single tryptophan at position 104 in the MBD, which is known from the NMR structure to be buried (4Wakefield R.I.D. Smith B.O. Nan X. Free A. Soteriou A. Uhrin D. Bird A.P. Barlow P.N. J. Mol. Biol. 1999; 291: 1055-1065Crossref PubMed Scopus (167) Google Scholar) and thus expected to have minimal solvent exposure. In a hydrophobic environment, tryptophan has a fluorescence emission maximum (λmax) of ∼330 nm. Loss of hydrophobic packing and exposure to the aqueous environment, as occurs upon complete thermal melting, results in a red shift of λmax to ∼355 nm accompanied by a large quenching of fluorescence intensity (42Carpenter, M. L., and Kneale, G. G. (1994) in Methods in Molecular Biology (Kneale, G. G., ed) pp. 313-325, Humana Press, Totowa, NJGoogle Scholar). Fig. 2a shows data collected at 25 and 75 °C for the full-length wild-type MeCP2 and proteins with four RTT-causing MBD mutations (Fig. 1), revealing the striking differences between the WT and the mutant proteins (Table 1). All except F155S and R106W had a λmax of ∼328 nm, consistent with strong hydrophobic packing of Trp-104. As expected, R106W had the highest fluorescence intensity, because it has two tryptophans and a λmax of 330 nm. Of more interest was the significant quenching of fluorescence in R133C and F155S, most likely the result of increased solvent exposure. F155S also showed an increase in λmax to ∼335 nm, suggesting that it is partially unfolded even at 25 °C. Indeed, spectra of F155S recorded at 5 °C showed a λmax close to the WT and a higher fluorescence intensity (not shown).TABLE 1Fluorescence emission and thermal unfolding parameters of full-length MeCP2 and MBDs in the presence and absence of methylated (Met) and, for wild type, unmethylated (Unmet) DNA. ND, not doneMeCP2 typeλmax at 25 °CFluorescence emission at λmax and 25 °CTm of protein onlyΔTm (mutant - WT)Tm of protein + DNADNA-induced ΔTm°C°C°C°CFull-length WT MeCP2328112,000 (100%)44.5, S.E. 0.263.1, S.E. 0.4 (Met)18.655.7, S.E. 0.5 (Unmet)11.2Full-length T158M329115,000 (103%)35.6, S.E. 0.3−8.948.4, S.E. 0.312.8Full-length R133C32882,000 (73%)38.0, S.E. 0.4−6.548.7, S.E. 0.210.7Full-length F155S33567,000 (60%)33.6, S.E. 0.4−10.945.3, S.E. 0.211.7Full-length R106W330149,000 (133%)44.8, S.E. 0.3+0.353.8, S.E. 0.39.0WT MBD32873,000 (100%)44.9, S.E. 0.154.3, S.E. 0.3 (Met)9.446.6, S.E. 0.6 (Unmet)1.7T158M MBD32961,000 (84%)39.1, S.E. 0.2−5.8NDR133C MBD32872,000 (99%)41.5, S.E. 0.3−3.4NDF155S MBD332.534,000 (47%)39.9, S.E. 0.1−5.0ND Open table in a new tab When the proteins were fully “melted” at 75 °C and Trp-104 maximally exposed, all showed a λmax of ∼350 nm and a much reduced intensity (Fig. 2a). The residual intensity of the 2-tryptophan R106W mutant at 75 °C was approximately twice that of the WT and other mutants (Fig. 2a). After heating to 85 °C and recooling, all proteins recovered ∼90% of the native fluorescence intensity at λmax (not shown), showing that heating did not induce aggregation and that the changes accompanying thermal melting were largely reversible. To examine the thermal stability of the wild-type and mutant proteins, fluorescence emission was recorded at 2.5 °C intervals from 5 to 85 °C. Plots of relative fluorescence intensity versus temperature yielded sigmoidal curves (Fig. 2b) indicative of cooperative unfolding. Temperatures resulting in 50% unfolding (Tm) obtained both from least square square fits of these curves or from the peak of the first derivative (Fig. 3a, inset) were essentially identical. Compared with WT MeCP2, the T158M, R133C, and F155S mutant proteins all showed a striking reduction in Tm, indicating an increased susceptibility to thermally induced unfolding (Fig. 2b, Table 1). The mutants F155S and R106W also showed considerable broadening of the slope of the melting transition (Fig. 2b), suggesting a decrease in the cooperativity of unfolding. To examine the effect of DNA binding on thermal, stability we selected a 45-bp segment of BDNF promoter DNA containing a single CpG and a nearby AT run known to bind MeCP2 in vivo (43Chen W.G. Chang Q. Lin Y. Meissner A. West A.E. Griffith E.C. Jaenisch R. Greenberg M.E. Science. 2003; 302: 885-889Crossref PubMed Scopus (1010) Google Scholar, 44Martinowich K. Hattori D. Wu H. Fouse S. He F. Hu Y. Fan G. Sun Y.E. Science. 2003; 302: 890-893Crossref PubMed Scopus (1169) Google Scholar) and that can be methylated as desired (45Klose R.J. Sarraf S.A. Schmiedeberg L. McDermott S.M. Stancheva I. Bird A.P. Mol. Cell. 2005; 19: 667-678Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). By comparing thermal unfolding in the presence and absence of DNA, it was possible to determine the effect of DNA on the stability of the protein. When exposed to methylated DNA, the wild type showed a dramatic 18.6 °C increase in Tm (Fig. 3a). A smaller increase (11.2 °C) in Tm was also observed with unmethylated DNA (Fig. 3a), in agreement with earlier findings of MeCP2-DNA binding in the absence of CpG methylation (6Adams V.H. McBryant S.J. Wade P.A. Woodcock C.L. Hansen J.C. J. Biol. Chem. 2007; 282: 15057-15064Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 19Nikitina T.N. Shi X. Ghosh R. Horowitz-Scherer R.A. Hansen J.C. Woodcock C.L. Mol. Cell. Biol. 2007; 27: 864-877Crossref PubMed Scopus (143) Google Scholar). The R133C, T158M, and F155S mutant proteins showed much smaller methylated DNA-induced increases in Tm as compared with the wild type (Fig. 3, b-d, Table 1). Thus, the binding of this small segment of DNA strongly stabilizes the wild-type MeCP2 against thermal melting but has a significantly weaker effect on the mutants, indicating a weaker binding affinity. The Structure of the MBD Is Altered in RTT-causing Mutants—We next examined the structural characteristics of the MBD alone. As shown in Fig. 4d, the WT MBD had a λmax similar to that of the full-length protein but a 35% reduction in fluorescence intensity a" @default.
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• W1970149098 title "Rett Syndrome-causing Mutations in Human MeCP2 Result in Diverse Structural Changes That Impact Folding and DNA Interactions" @default.
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