Defining potential roles of Pb(2+) in neurotoxicity from a calciomics approach

Abstract

Metal ions play crucial roles in numerous biological processes, facilitating biochemical reactions by binding to various proteins. An increasing body of evidence suggests that neurotoxicity associated with exposure to nonessential metals (e.g., Pb(2+)) involves disruption of synaptic activity, and these observed effects are associated with the ability of Pb(2+) to interfere with Zn(2+) and Ca(2+)-dependent functions. However, the molecular mechanism behind Pb(2+) toxicity remains a topic of debate. In this review, we first discuss potential neuronal Ca(2+) binding protein (CaBP) targets for Pb(2+) such as calmodulin (CaM), synaptotagmin, neuronal calcium sensor-1 (NCS-1), N-methyl-d-aspartate receptor (NMDAR) and family C of G-protein coupled receptors (cGPCRs), and their involvement in Ca(2+)-signalling pathways. We then compare metal binding properties between Ca(2+) and Pb(2+) to understand the structural implications of Pb(2+) binding to CaBPs. Statistical and biophysical studies (e.g., NMR and fluorescence spectroscopy) of Pb(2+) binding are discussed to investigate the molecular mechanism behind Pb(2+) toxicity. These studies identify an opportunistic, allosteric binding of Pb(2+) to CaM, which is distinct from ionic displacement. Together, these data suggest three potential modes of Pb(2+) activity related to molecular and/or neural toxicity: (i) Pb(2+) can occupy Ca(2+)-binding sites, inhibiting the activity of the protein by structural modulation, (ii) Pb(2+) can mimic Ca(2+) in the binding sites, falsely activating the protein and perturbing downstream activities, or (iii) Pb(2+) can bind outside of the Ca(2+)-binding sites, resulting in the allosteric modulation of the protein activity. Moreover, the data further suggest that even low concentrations of Pb(2+) can interfere at multiple points within the neuronal Ca(2+) signalling pathways to cause neurotoxicity.

Fig. 1

Ca²⁺ and Pb²⁺ targeted proteins involved directly and indirectly in neurotoxicity and calcium homeostasis are illustrated. The Ca²⁺ gradient is maintained at nM range intracellularly and at a mM range extracellularly in neurons. The black arrows represent the influx and the yellow arrows represent the outflux of divalent metal ions in the system. A general activity profile of receptor proteins is shown by a sigmoid and a bell curve as a result of Ca²⁺ and Pb²⁺ ions, respectively. Pb²⁺ is known to activate Ca²⁺ associated neuronal proteins at low concentrations and deactivate at higher concentrations, as characterized by a bell curve.

Fig. 2

(A) Proposed allosteric binding site for Pb²⁺ in human Ca²⁺-bound CaM (PDB ID 4bw8) in carboxyl-rich region of central helix comprising residues 78-84 (DTDSEEE). (B) Pb²⁺-bound human CaM (PDB ID 4bw8) (C) Human Ca²⁺-bound NCS-1 (PDB ID 4guk) with four EF-Hand sites similar to those observed in CaM (D) Pb²⁺ bound in site EF-IV of CaM (PDB ID 2v01). (E) Comparison of Ca²⁺- and Pb²⁺-binding characteristics with respect to the pentagonal plane. D_Lig-Ca and D_Lig-Pb, indicate distances between oxygen ligands and ions. C-Lig-Ca and C-Lig-Pb represent angles ϕ formed between the carbon atoms, ligands and the ions. D_Ca and D_Pb indicate ion distance above the pentagonal plane formed by C^γ, O^δ1 and O^δ2. (F) Ca²⁺ bound in site EF-IV of CaM (PDB ID 3cln). Figs. 2A, B, C, D, and F rendered with Chimera (24).

Fig. 3

Proposed mechanism for Pb²⁺ toxicity in CaM. Path A illustrates normal activity associated with binding of Ca²⁺. Ca²⁺ first cooperatively binds the two higher affinity sites EF-III and EF-IV, followed by cooperative binding in sites EF-I and EF-II. The Ca²⁺ loaded protein then interacts with ligand molecules. In Path B, Pb²⁺ binds in a single higher affinity site in the C-terminal domain, while the remaining sites may bind Pb²⁺ with equivalent affinity. Binding may not occur cooperatively. At lower concentration of Pb²⁺, CaM is activated and functions as if bound with Ca²⁺. In either the Ca²⁺ or Pb²⁺ loaded states, an increase in the concentration of Pb²⁺ results in Pb²⁺ binding to a secondary site in the central helix, altering the conformation of the protein and allosterically inhibiting the activity of the protein.