Calcium-regulated DNA Binding and Oligomerization of the Neuronal Calcium-sensing Protein, Calsenilin/DREAM/KChIP3

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 (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 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 Ca²⁺-free state and dissociates into dimers at saturating Ca²⁺ levels.