Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.

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

doi:10.1074/jbc.m105842200

Abstract

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.

Highlights

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
Isothermal titration calorimetry and dynamic light scattering indicate that the Ca2؉-free protein tetramer binds endothermically (⌬H ‫ ؍‬؉25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes
Like other members of the recoverin family (Fig. 1), the first EF-hand from the N terminus (EF-1) of DREAM contains substitutions (Cys-104 and Pro-105) that are expected to disrupt the structure of this binding loop and prevent Ca2ϩ binding to EF-1, as seen in the crystal structures of recoverin [20], neurocalcin [21], and frequenin [22]

Summary

Introduction

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. The samples for the experiments examining concentration dependence of the molecular mass were prepared by dilution of the protein stock solutions previously dialyzed against 10 mM Tris-HCl, pH 7.4, 10 mM LDAO, 1 mM DTT, and 1 mM EDTA or 5 mM CaCl2 for apo- and Ca2ϩ-DREAM, respectively.

Results

Conclusion