Ca2+ and Mg2+ Induced Conformational Changes in Downstream Regulatory Antagonist Modulator (DREAM) Measured by Extrinsic Hydrophobic Probes

Abstract

DREAM (downstream regulatory element antagonist modulator) is a multifunctional calcium sensor protein involved in regulation of gene expression and K-channel activity in neuronal cells. Here we present spectroscopic studies of the Ca2+ triggered conformational transition in DREAM using the ANS extrinsic hydrophobic probes 1,8-anilinonapthalene and 2,6-anilinonapthalene. Fluorescence emission spectra of the ANS-DREAM complexes show an increase in ANS emission intensity for Ca2+ and Ca2+Mg2+ bound DREAM consistent with an overall decrease in surface polarity due to the Ca2+ binding. From the titration studies the ANS dissociation constants were determined to be: Kd1,8-ANS = 145±15 μM for the apoform and Kd1,8-ANS = 45±2 μM for Ca2+DREAM. Two binding sites for both ANS isomers were identified in apo/Mg2+ and Ca2+/Mg2+Ca2+ protein using fluorescence lifetime measurements. We propose that one binding site is partially solvent exposed and exhibits a shorter lifetime, τ1,8-ANS = 5.8 ns and τ2,6-ANS = 6.8 ns, whereas the second site is buried within the protein matrix with τ1,8-ANS = 16.1 ns and τ2,6-ANS = 10.9 ns. Computational simulation of ligand binding support the two site binding model with one site located between EF-3/4 and the second site near Trp169 and the entering helix of EF-3. Using fluorescence anisotropy decay two correlation times were resolved for apoprotein and Ca2+ bound DREAM. We assign the longer correlation time to a global motion of the protein (Φapo/Mg2+ = 55 ns and ΦCa2+/Mg2+Ca2+ = 26 ns) and the second time to a local motion of the C-terminal domain (Φapo/Mg2+ = 13 ns and ΦCa2+/Mg2+Ca2+ = 6 ns). The 55 ns correlation time is consistent with apo and Mg2+DREAM being in the tetrameric form whereas Ca2+ binding to apo or Mg2+ DREAM promotes formation of DREAM dimer.