Abstract
DREAM, an EF-hand protein, associates with and modulates the activity of presenilins and Kv4 potassium channels in neural and cardiac tissues and represses prodynorphin and c-fos gene expression by binding to DNA response elements in these genes. Information concerning the metal-binding properties of DREAM and the consequences of metal binding on protein structure are important in understanding how this protein functions in cells. We now show that DREAM binds 1 mol of calcium/mol of protein with relatively high affinity and another 3 mol of calcium with lower affinity. DREAM binds 1 mol of magnesium/mol of protein. DREAM, pre-loaded with 1 mol of calcium, binds 1 mol of magnesium, thus demonstrating that the magnesium-binding site is distinct from the high affinity calcium-binding site. Analysis of metal binding to mutant DREAM protein constructs localizes the high affinity calcium-binding site and the magnesium-binding site to EF-hands 3 or 4. Binding of calcium but not magnesium changes the conformation, stability, and alpha-helical content of DREAM. Calcium, but not magnesium, reduces the affinity of apo-DREAM for specific DNA response elements in the prodynorphin and c-fos genes. We conclude that DREAM binds calcium and magnesium and that calcium, but not magnesium, modulates DREAM structure and function.
Highlights
DREAM, an EF-hand protein, associates with and modulates the activity of presenilins and Kv4 potassium channels in neural and cardiac tissues and represses prodynorphin and c-fos gene expression by binding to DNA response elements in these genes
Because familial forms of Alzheimer’s disease are often associated with mutations in the presenilin (PS)1 1 and 2 genes (9 –18), and mutations in PS1 are associated with a relative increase in A42 in amyloid plaques seen in this disease, it is conceivable that DREAM modulation of PS function may be altered in this disease
DREAM is a member of a family of proteins that interact with the amino terminus of calcium-activated Kv4 potassium channels (8)
Summary
General—Protein amino acid composition, protein amino-terminal and carboxyl-terminal sequencing, and DNA sequencing were carried out as described (26 –28). Oligonucleotide synthesis (29) was performed using an Applied Biosystems DNA/oligonucleotide synthesizer (Applied Biosystems, Foster City, CA). UV spectra of proteins and nucleic acids were recorded using a model DU-70 or DU-640 Beckman spectrophotometer (Beckman Instruments, Fullerton, CA). Protein concentrations were determined by amino acid analysis, by the Bradford method (30), or by measuring UV absorbance using the molar absorptivity of DREAM at 281 nm of 29.6 Ϯ 0.3 mMϪ1 cmϪ1 obtained by quantitative determination of nitrogen by the indophenol blue method (31). Biosynthesis of DREAM and DREAM Mutant Proteins—We synthesized the following DREAM proteins by methods detailed below for the full-length DREAM protein.
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