Characterization of the Role of Individual EF-Hands in Dream in Modulating Conformational Dynamics, Oligomeric States, and Interaction with DNA

Abstract

Downstream Regulatory Element Antagonistic Modulator (DREAM) carries four calcium binding motifs (EF-hand) of which EF-3 and EF-4 bind to Ca2+ with high affinity, whereas EF-2 preferentially binds to Mg2+ and EF-1 is inactive. In neuronal cells, DREAM serves as a gene transcriptional repressor of dynorphin, an endogenous ligand, to regulate pain transmission by controlling kappa receptor activation. Recent findings have shown that tetrameric ApoDREAM binds to DNA with a high affinity, whereas association of Ca2+ abolishes the interaction with DNA. Despite mechanisms of interactions between DREAM and DNA have been proposed, it remains unclear which EF-hand in DREAM triggers tetramer dissociation and thus controls DNA affinity. In this study, a combination of fluorescence steady-state, time-resolved anisotropy, circular dichroism (CD), and isothermal titration calorimetry (ITC) techniques were employed to investigate the role of individual EF-hands. Here, we showed that Mg2+ binding to EF-2 does not affect the tertiary and secondary structures of DREAM. Ca2+ binding to EF-3 induces conformational changes in DREAM evidenced by a blue-shift and a decrease in emission intensity of the single tryptophan, whereas the association of Ca2+ to EF-4 does not disturb the DREAM structure. We also showed that elimination of Mg2+ binding to DREAM results in dimerization in Apo form and increase of the dimeric fraction in Ca2+ form. In contrast, abolishment of Ca2+ binding to EF-4 results in a high fraction of DREAM tetramer (60-75%) in Apo, Mg2+, Ca2+, and Ca2+Mg2+ bound forms. Finally, ITC results showed that ApoDREAM wild-type endothermically (ΔH=+32 kcal/mol) binds to prodynorphin DRE oligomer with a Kd of 0.5 µM, whereas a weak interaction was observed in the Ca2+ form (Kd∼200 µM). Interactions of DREAM mutants with DNA will also be discussed.