TY - JOUR
T1 - Calcium-regulated DNA Binding and Oligomerization of the Neuronal Calcium-sensing Protein, Calsenilin/DREAM/KChIP3
AU - Osawa, Masanori
AU - Tong, Kit I.
AU - Lilliehook, Christina
AU - Wasco, Wilma
AU - Buxbaum, Joseph D.
AU - Cheng, H. Y.Mary
AU - Penninger, Josef M.
AU - Ikura, Mitsuhiko
AU - Ames, James B.
PY - 2001/11/2
Y1 - 2001/11/2
N2 - Calsenilin/DREAM/KChlP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca2+ binding measurements indicate that the protein binds 3 Ca2+ with a dissociation constant of 14 μM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca 2+-bound protein exists as a dimer at protein concentrations lower than 150 μM and forms a tetramer at concentrations above 200 μM. The Ca2+-free protein is a tetramer in the concentration range 20-450 μM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca2+-free protein tetramer binds endothermically (δH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (Kd) of 75 nM, and the other three bind more weakly (Kd = 640 nM). No significant DNA binding was observed for the Ca2+-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca 2+-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (Kd = 200 nM) in a Ca 2+-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca2+-free state and dissociates into dimers at saturating Ca2+ levels.
AB - Calsenilin/DREAM/KChlP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca2+ binding measurements indicate that the protein binds 3 Ca2+ with a dissociation constant of 14 μM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca 2+-bound protein exists as a dimer at protein concentrations lower than 150 μM and forms a tetramer at concentrations above 200 μM. The Ca2+-free protein is a tetramer in the concentration range 20-450 μM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca2+-free protein tetramer binds endothermically (δH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (Kd) of 75 nM, and the other three bind more weakly (Kd = 640 nM). No significant DNA binding was observed for the Ca2+-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca 2+-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (Kd = 200 nM) in a Ca 2+-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca2+-free state and dissociates into dimers at saturating Ca2+ levels.
UR - http://www.scopus.com/inward/record.url?scp=0035798666&partnerID=8YFLogxK
U2 - 10.1074/jbc.M105842200
DO - 10.1074/jbc.M105842200
M3 - Article
C2 - 11535596
AN - SCOPUS:0035798666
SN - 0021-9258
VL - 276
SP - 41005
EP - 41013
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 44
ER -