Abstract
Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensor proteins that are expressed in the central nervous system including the hippocampus and the neural cortex. DREAM interacts with a variety of intracellular partners, and is involved in regulating gene expression, kinetics of potassium channels, calcium homeostasis, and enzymatic activity of presenilin; it was proposed to be involved in several physiological processes such as memory, learning and pain sensitivity; and linked to pathological conditions such as Alzheimer's and Huntington's disease. Previous studies have indicated that calcium association to DREAM leads to the formation of a Ca2+ bound dimer, while in the apo state a monomer-tetramer equilibrium was observed. In order to better understand Ca2+ triggered dimerization process, we prepared a chimeric version of DREAM (DREAM-NCS1) with Arg 200/Arg 220, and Leu 158/Leu 159 replaced by residues corresponding to the analogues residues in NCS-1, with the aim to remove salt bridges and hydrophobic interactions stabilizing DREAM dimer. DREAM-NCS1 properties were investigated using molecular dynamic simulations and spectroscopic approaches. Fluorescence emission data shows a distinct reorganization of W169 environment upon Ca2+ binding to DREAM-NCS1, with respect to WT, whereas enhancement in the emission of DREAM-NCS: 1,8-ANS complex points towards an increased exposure of the hydrophobic sites in the chimeric protein. Furthermore, low polarization values for DREAM-NCS1 bound to peptides mimicking site 1 and site 2 in the T1 domain of Kv channels are consistent with the lower affinity of the chimeric protein for each site as well as with the presence of the DREAM-NCS-1 monomeric form in the presence of Ca2+. These data points towards the role of both, hydrophobic interactions and salt bridges in stabilizing the dimeric form of the protein as well as in propagating allosteric signals.
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