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 Ca2+ binding measurements indicate that the protein binds 3 Ca2+ with a dissociation constant of 14 muM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca2+-bound protein exists as a dimer at protein concentrations lower than 150 muM and forms a tetramer at concentrations above 200 muM. The Ca2+-free protein is a tetramer in the concentration range 20-450 muM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca2+-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 Ca2+-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca2+-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (K-d = 200 nM) in a Ca2+-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.