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J. Biol. Chem., Vol. 283, Issue 29, 20523-20534, July 18, 2008

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Rett Syndrome-causing Mutations in Human MeCP2 Result in Diverse Structural Changes That Impact Folding and DNA Interactions^*

Rajarshi P. Ghosh, Rachel A. Horowitz-Scherer, Tatiana Nikitina, Lila M. Gierasch^¶, and Christopher L. Woodcock¹

From the Department of Biology, Program in Molecular and Cellular Biology, and ^¶Department of Biochemistry and Molecular Biology and Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003

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 -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.

Received for publication, April 21, 2008 , and in revised form, May 21, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM070897 (to C. L. W.) and GM027616 (to L. M. G.). This work was also supported by the International Rett Foundation (to C. L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Biology Dept., University of Massachusetts, Amherst, MA 01003. Tel.: 413-545-2825; E-mail: chris{at}bio.umass.edu.

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