The overall objectives are to elucidate the structure and mechanism of action of a Ca2+-sensitive transcriptional repressor protein named, DREAM (Downstream Regulatory Element Antagonist Modulator). DREAM, a member of the neuronal calcium sensor subclass of the EF-hand super family, is expressed in the brain where it binds to specific DNA sequences downstream of the promoter region (DRE, Downstream Regulatory Element) and blocks transcription of downstream targets only at low Ca2+ levels. DREAM has been shown to suppress the transcription of prodynorphin and c-fos genes at basal Ca2+ levels, but does not suppress transcription at elevated Ca2+ levels caused by cell stimulation or injury. The importance of DREAM as a calcium-sensitive transcriptional repressor has been demonstrated in DREAM-deficient mice that exhibit the very striking phenotype of ongoing analgesia due to upregulated expression of prodynorphin. Importantly, the DREAM knockout mice do not exhibit any motor or behavioral abnormalities. Hence, DREAM regulates pain transmission by controlling prodynorphin expression and represents an attractive therapeutic opportunity for managing pain. The atomic-level structural characterization of DREAM and its Ca2+-regulated interaction with target DNA are crucial for the development of therapeutics designed to eliminate pain by specifically targeting DREAM.
The specific aims are 4-fold:
The first aim i s to determine the structure of the C-terminal calcium-binding domain of DREAM in solution by nuclear magnetic resonance (NMR). The structure is important for understanding how calcium-induced conformational changes within the EF-hand motifs control the binding of DNA targets.
The second aim i s to measure the energetics and kinetics of DREAM binding to calcium and DNA targets. The thermodynamics and kinetics of binding will augment the structural data in the rational design of drugs that target DREAM and block pain.
The third aim i s to determine the active-site amino acid residues of DREAM that control binding of DNA targets. Site directed mutagenesis studies will identify important amino acid residues and structural interactions of the target complex and may reveal structural determinants important for drug design.
The fourth aim i s to obtain high quality protein crystals of DREAM that will then be used to solve the atomic resolution structure of the protein-DNA complex by x-ray crystallography.
