Pb2+ Binds to Downstream Regulatory Element Antagonist Modulator (DREAM) and Modulates Its Interactions with Binding Partners: A Link between Neuronal Calcium Sensors and Pb2+ Neurotoxicity.

Abstract

Pb2+ exposure leads to diverse neurological disorders; however, the mechanism of Pb2+-induced neurotoxicity is not clearly understood. Here we demonstrate that Pb2+ binds to EF-hands in apo-DREAM (downstream regulatory element antagonist modulator) with a lower equilibrium dissociation constant ( Kd = 20 ± 2 nM) than Ca2+ ( Kd = 1 μM). Based on the Trp169 emission and CD spectra, we report that Pb2+ association triggers changes in the protein secondary and tertiary structures that are analogous to those previously observed for Ca2+-bound protein. 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-anilinonaphthalene-8-sulfonic acid (1,8-ANS). Pb2+ association with DREAM also modulates interactions between DREAM and its intracellular partners as evident from the fact that Pb2+-bound DREAM associates with peptide-based model systems, presenilin-1 helix-9 "PS1HL9" KV4.3(70-90) "site-2" and KV4.3(2-22) "site 1". Namely, dissociation constants for Pb2+-bound DREAM interaction with PS1HL9 ( Kd = 2.4 ± 0.1 μM), site-2 ( Kd = 11.0 ± 0.5 μM) and site 1 ( Kd = 5.0 ± 0.6 μM) are nearly identical to those observed for Ca2+ bound DREAM. Isothermal titration calorimetry data reveal that Pb2+ binds to two high-affinity sites in Ca2+ bound DREAM with the overall apparent constant of 4.81 ± 0.06 μM and its binding to Ca2+ bound DREAM is entropy-driven. Taking into account the structural and sequence similarity between DREAM and other neuronal calcium sensor (NCS) proteins, these results strongly indicate that DREAM and possibly other NCS proteins bind Pb2+ with a higher affinity than that for Ca2+ and Pb2+ interactions with NCS proteins can contribute to Pb2+-induced neurotoxicity.