Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.
Calsenilin/DREAM/KChIP3, 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 Ca(2+) binding measurements indicate that the protein binds 3 Ca(2+) with a dissociation constant of 14 microM 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 microM and forms a tetramer at concentrations above 200 microM. The Ca(2+)-free protein is a tetramer in the concentration range 20-450 microM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca(2+)-free protein tetramer binds endothermically (DeltaH = +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 (K(d)) of 75 nM, and the other three bind more weakly (K(d) = 640 nM). No significant DNA binding was observed for the Ca(2+)-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 (K(d) = 200 nM) in a Ca(2+)-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca(2+)-free state and dissociates into dimers at saturating Ca(2+) levels.
Osawa
M
M
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA.
Tong
K I
KI
Lilliehook
C
C
Wasco
W
W
Buxbaum
J D
JD
Cheng
H Y
HY
Penninger
J M
JM
Ikura
M
M
Ames
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001704:Biopolymers; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D005786:Gene Expression Regulation; D006801:Humans; D051605:Kv Channel-Interacting Proteins; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012097:Repressor Proteins; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D001704:Biopolymers; D002135:Calcium-Binding Proteins; D017931:DNA Primers; C496749:KCNIP3 protein, human; D051605:Kv Channel-Interacting Proteins; D012097:Repressor Proteins; D004247:DNA; D002118:Calcium