Structural characterization of metal and DNA binding to DREAM protein, a calcium sensing transcriptional repressor in pain modulation
Valiveti, Aswani Kumar
Ames, James B
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DREAM (Downstream Regulatory Element Antagonistic Modulator) is the first reported calcium binding protein that directly binds to DNA and affects gene expression. It belongs to recoverin sub-family of neuronal calcium sensing proteins that form part of EF- hand super-family. Knockout mice, devoid of DREAM protein, exhibit ongoing analgesia due to upregulated expression of the prodynorphin gene. DREAM binds to a specific DNA silencing element in the prodynorphin promoter called Downstream Regulatory Element (DRE) and serves as a transcriptional repressor under basal resting conditions. Neuronal stimulation leads to a rise in nuclear calcium, causing Ca++-bound DREAM to dissociate from DNA and result in derepression of prodynorphin expression. Thus, DREAM may serve as a potentially specific target for effective pain management. Understanding the structural determinants of metal binding and DNA recognition by DREAM may help in the future rational design of analgesic drugs. In this thesis, the calcium and magnesium binding properties of DREAM were studied by performing isothermal titration calorimetry (ITC) analysis on wild-type DREAM protein and mutants that have each of the functional EF hands disabled for calcium binding. Our results show that DREAM binds two Ca++ ions and one Mg++ ion with high affinity. Mg++-bound DREAM is stable as a monomer and Ca++-bound DREAM exists as a dimer. Calcium binding stabilizes the protein structure and promotes dimerization to control calcium-sensitive recognition of DNA targets. Next, we attempted to map the DNA binding residues in DREAM and its oligomerization interface using biophysical and functional characterization of chimeric protein constructs generated by swapping specific EF-hand motifs of DREAM protein with those of recoverin. Nuclear magnetic resonance (NMR), gel-filtration and electrophoretic mobility shift assays were employed for this purpose. The results from this study suggest that both the amino and carboxy terminal halves of the protein are important for DNA binding and dimerization of DREAM protein. All four EF-hands in DREAM appear to participate in the recognition of DNA.