1. Zoghbi et al., Postnatal Neurodevelopmental Disorders: Meeting at the Synapse?, Science 302:826-830 (2003).
2. Ho et al., MeCP2 binding to DNA Depends upon Hydration at Methyl-CpG, Mol. Cell 29:525-531 (2008).
3. Guy et al., Reversal of Neurological Defects in a Mouse Model of Rett Syndrome, Science (2007).
4. Marchetto et al., A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells, Cell 143:527-539 (2010).
www.rcsb.org Rutgers Center for Structural Biology. Database of biological structures.
www.ncbi.nlm.nih.gov/omim/ Online Mendelian Disorders in Man. Database of medical and genetic information on inherited human diseases.
Concise answers to the Study Guide questions and molecular images resulting from the modeling exercises are available on the Proteopedia website. Simply type in ‘autism’ in the search bar.
1. Rett Syndrome. Describe the sequence of events leading to the discovery in 1999 by Huda Zoghbi that mutations in MeCP2 cause Rett Syndrome. Why is this disease almost exclusively found in girls? What does it mean that this syndrome is caused by mosaic expression of mutant copies of the X-linked mecp2 gene? Does this explain the phenotypic variability seen in Rett Syndrome (the range of mild to severe cases)? What is the significance of the observation that mecp2 expression steadily increases and eventually affects all major brain regions? What other diseases appear to be due to dysfunctional synapses? What are the functions of the proteins implicated (FMR1, UBE3A, NLGN, NRXN, GABAR3, SHANK3, PTEN, TSC1/TSC2)?
2. MeCP2. What is the normal biological function of MeCP2? Describe the organization of MeCP2 into its two major functional domains. What is meant by ‘chromatin remodeling’ and what is the relevance for understanding the function of MeCP2? What is ‘genomic imprinting’? Describe how the MeCP2 DNA-binding sequence used in the crystallization of the complex was designed. Draw the chemical structure of a methyl-GC base pair.
3. Mouse model. Describe the mouse model engineered by Adrian Bird and colleagues that was used to show that, in principle, the neurological effects in Rett Syndrome can be reversed. Give a brief description of long-term potentiation (LTP)? (Do not attempt to describe in detail the electrophysiological results that enable Bird and colleagues to make the claim of phenotypic reversibility. This will come later when we study neurotransmission.). Although this is not a cure, what hope can be offered to families affected by this disorder?
4. MeCP2-DNA Structure. How did Ho et al. select the length and sequence of the DNA used in their crystallization trials? In the MBD-DNA structure what is surprising about the contacts involved in methyl CpG di-nucleotide recognition? What amino acid side-chains make direct contact with the methyl group? What specific H-bond interactions do these side-chains have with DNA? Describe the salt-bridges that stabilize the positions of these side-chains? Describe the crucial role played by five specific water molecules in the network of interactions involved in MeCP2 binding to its target site.
Using SPDBV or RASMOL to construct an image(s) of the specific contacts made by side-chains to the methyl groups on the two cytosines. You have a lot of flexibility in your choice of representations (space-filling for side-chains, wire-model for DNA, ribbons for protein atoms not involved, etc.).
One of the most frequently mutated sites in Rett Syndrome is T158M. Does the crystal structure offer an explanation for the disruptive effect this mutation? Explain how the R106W mutation might affect the function of MeCP2. Extract the structure of the Asx-TS motif and create different images in SPDBVor RASMOL in order to elucidate the important intra-chain interactions in this unusual structure (i.e. reproduce figure 4 adding your own artistic flair). What interactions of the T158 side-chain appear to direct the course of the polypeptide chain C-terminal to take on the structure of this motif? Study carefully the precise role played by V159 in the protein-DNA interaction. How is the structure of DNA affected by runs of AT base pairs flanking meCpG sites? How does this contribute to the specificity of the MBD-DNA binding interaction? Produce an image that shows the interaction between V159 and the minor groove of the target DNA and discuss its proximity to the methyl cytosine binding region. Produce a Ramachandran diagram showing the phi/psi angles for the Asn-TS substructure.
5. The use of Inducible Pluriopotent Stem Cells (IPSCs) to discover therapeutic agents. How are IPSCs produced? What makes them potentially so valuable for drug discovery? What functional properties of IPSC’s prepared from the fibroblasts of Rett patients would be important to establish if they were to be useful for drug discovery? Do the IPSC’s described by Marchetto et al. display normal patterns of X-inactivation? What is the rationale behind each of the two drug leads tested in the Marchetto paper?
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