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

Masanori Osawa, Kit I. Tong, Christina Lilliehook, Wilma Wasco, Joseph D. Buxbaum, H. Y.Mary Cheng, Josef M. Penninger, Mitsuhiko Ikura, James B. Ames

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109 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)41005-41013
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number44
DOIs
Publication statusPublished - 2001 Nov 2
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Osawa, M., Tong, K. I., Lilliehook, C., Wasco, W., Buxbaum, J. D., Cheng, H. Y. M., Penninger, J. M., Ikura, M., & Ames, J. B. (2001). Calcium-regulated DNA Binding and Oligomerization of the Neuronal Calcium-sensing Protein, Calsenilin/DREAM/KChIP3. Journal of Biological Chemistry, 276(44), 41005-41013. https://doi.org/10.1074/jbc.M105842200