Lead tightly associates with neuronal calcium sensor (NCS) protein DREAM and promotes structural changes analogous to calcium bound DREAM.

Samiol Azam, Jaroslava Miksovska

Florida International University, Miami, Fl, USA

Downstream regulatory element antagonist modulator (DREAM), is a 29kDa Ca²⁺ binding protein that controls several neurological processes including gene expression, apoptosis, and modulation of K_v4 voltage channels. Here we demonstrate that Pb²⁺ binds to EF-hands in DREAM with and equilibrium affinity higher that that determined for Ca²⁺. Pb²⁺ association triggers changes in the secondary and tertiary structure of the protein that are analogous to those observed in Ca²⁺ bound DREAM based on Trp 169 emission data and CD spectra. Namely, Pb²⁺ binding to DREAM leads to decrease of Trp 169 emission intensity and decrease in CD signal at 220nm. The hydrophobic cavity in the C-terminal domain of DREAM is solvent exposed in the presence of Pb²⁺ as determined using a hydrophobic probe 1, 8-ANS. The K_d values for 1,8-ANS binding to Ca²⁺ and Pb²⁺ bound DREAM were found to be similar; 73±10 μM and 103 ± 10 µM, respectively. Pb²⁺ binding to DREAM also modulates DREAM interactions with intracellular partners. For example, titrations of presenilin-1  with DREAM in the presence of Ca²⁺ or Pb²⁺ showed that Pb²⁺ bound DREAM has a similar affinity for presenilin-1 (K_d= 2.44±0.19 µM) as Ca²⁺ bound DREAM(K_d= 6.23 ± 1.60 µM). The impact of Pb²⁺ association on DREAM conformational dynamics was proved in time-resolved fluorescence studies and thermodynamics parameters for Pb²⁺ association to EF-hands were obtained using isothermal titration calorimetry (ITC). Those results indicate that DREAM and likely other neuronal calcium sensors may contribute to lead-induced toxicity.