The metal-binding properties of DREAM: evidence for calcium-mediated changes in DREAM structure.
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.
Craig
Theodore A
TA
Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Benson
Linda M
LM
Venyaminov
Sergei Yu
SY
Klimtchuk
Elena S
ES
Bajzer
Zeljko
Z
Prendergast
Franklyn G
FG
Naylor
Stephen
S
Kumar
Rajiv
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D051605:Kv Channel-Interacting Proteins; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D051605:Kv Channel-Interacting Proteins; D012097:Repressor Proteins; D008274:Magnesium; D002118:Calcium