Abstract
Downstream Regulatory Element Antagonist Modulator (DREAM)/Calsenilin/KchIP3 is a multifunctional calcium binding protein that belongs to the EF-hand branch of Neuronal Calcium Sensor family. DREAM associates to Downstream Regulatory Element (DRE) of prodynorphin and c-fos genes and blocks their transcription in a calcium-regulated manner. Other molecular functions of DREAM involve proteolytic processing of presenilins, modulation of A-type current of potassium channels, and regulation of neuronal apoptosis. Previously, we have studied Mg2+ and Ca2+ induced changes in structure and dynamics of DREAM and its C-terminal domain. Recently, we have investigated the contribution of individual EF hands (EF-2, EF-3, and EF-4) to Ca2+ triggered conformational transition in DREAM by characterizing fluorescence properties of D150N, E186Q, and E234Q mutants. The tryptophan 169 emission and lifetime properties are strongly influenced by Ca2+ association to EF-3 whereas the ligand association to EF-2 and/or EF-4 has a minor impact on Trp fluorescence suggesting that Ca2+ association to EF-3 is crucial to induced structural changes within the hydrophobic pocket between EF-2 and EF-3. In addition, association of 25-mer oligonucleotide of prodynorphin gene to DREAM was characterized using ITC. DNA association to DREAM is temperature dependent. ApoDREAM strongly binds to DynDRE at 35oC (Kd1 =206 nM, Kd2 = 24 µM) whereas weaker interactions were observed at at 25oC). Very weak binding with Kd = 91 µM and Kd = 200 µM was observed for DREAM-DynDRE association in the presence of Mg2+ and Ca2+, respectively. These results support the mechanism that ApoDREAM strongly binds to DynDRE to block prodynorphin gene transcription and the DREAM-DynDRE complex reversibly dissociates upon Ca2+ binding.
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