Metal toxicity and opportunistic binding of Pb2+ in proteins

Abstract

Lead toxicity is associated with various human diseases. While Ca2+ binding proteins such as calmodulin (CaM) are often reported to be molecular targets for Pb2+-binding and lead toxicity, the effect of Pb2+ on the Ca2+/CaM regulated biological activities cannot be described by the primary mechanism of ionic displacement (e.g., ionic mimicry). The focus of this study was to investigate the mechanism of lead toxicity through binding differences between Ca2+ and Pb2+ for CaM, an essential intracellular trigger protein with two EF-Hand Ca2+-binding sites in each of its two domains that regulates many molecular targets via Ca2+-induced conformational change. Fluorescence changes in phenylalanine indicated that Pb2+ binds with 8-fold higher affinity than Ca2+ in the N-terminal domain. Additionally, NMR chemical shift changes and an unusual biphasic response observed in tyrosine fluorescence associated with C-terminal domain sites EF-III and EF-IV suggest a single higher affinity Pb2+-binding site with a 3-fold higher affinity than Ca2+, coupled with a second site exhibiting affinity nearly equivalent to that of the N-terminal domain sites. Our results further indicate that Pb2+ displaces Ca2+ only in the N-terminal domain, with minimal perturbation of the C-terminal domain, however significant structural/dynamic changes are observed in the trans-domain linker region which appear to be due to Pb2+-binding outside of the known calcium-binding sites. These data suggest that opportunistic Pb2+-binding in Ca2+/CaM has a profound impact on the conformation and dynamics of the essential molecular recognition sites of the central helix, and provides insight into the molecular toxicity of non-essential metal ions.

Defining potential roles of Pb2+in neurotoxicity from a calciomics approach

“…If this latter hypothesis were correct, it would help to explain the biphasic response of CaM activation to Pb 2+ , where trace concentrations can activate the protein, which is subsequently deactivated with increasing concentration. The study further noted that Pb 2+ did not produce conformational changes when Pb 2+ was introduced to Ca 2+ -loaded CaM, despite differences in binding affinity values, which suggests that Pb 2+ either displaces Ca 2+ without disrupting the structure, or Pb 2+ fails to displace Ca 2+ , but can deactivate the protein through opportunistic binding in a secondary site (123). In the latter case, it is possible that Pb 2+ may bind opportunistically to regions of the protein outside of the normal metal binding sites, such as the carboxyl-rich central helix of CaM (Fig.…”

“…The compact folding observed in the crystalline structure of Pb 2+ -bound CaM suggested a potential allosteric binding site for Pb 2+ in this region, and results of structural analyses identified the carboxyl-rich region from residues 78–84 as a strong candidate for binding of Pb 2+ (Fig. 2A) (123). …”

Pb(II) Disruption of Synaptic Activity Through Ca(II)- and Zn(II)-binding Proteins

“…To address these questions, a follow-up study was conducted that compared structural differences and binding affinities for Pb 2+ and Ca 2+ bound to CaM [84] . [85,86] , and with a related study by Dowd et al which reported that occupancy of Pb 2+ in Ca 2+ -binding sites may activate a protein, mimicking the presence of Ca 2+ , but may also result in a more compact conformation of the protein [87] .…”

“…These results suggest that, at lower concentrations, Pb 2+ mimics Ca 2+ by ionic replacement in structured Ca 2+ -binding sites, which would inappropriately activate the protein. Conversely, at higher concentrations, Pb 2+ appears to interact with CaM through a different mechanism involving opportunistic binding outside of the known Ca 2+ -binding sites, or in auxiliary binding sites for Zn 2+ and Mg 2+ , resulting in allosteric inhibition of protein activity [84] . Thus, CaM likely represents an important participant in Pb 2+ neurotoxicity across a range of Pb 2+ concentrations, and due to its ubiquitous presence across a multitude of functions regulated by the Ca 2+ -signaling network.…”

Heavy Metals in Sewage Treated Effluents: Pollution and Microbial Bioremediation from Arid Regions

“…It is one of the non-essential elements with no role in human metabolism, with children being more affected than adults [59]. Lead replaces calcium in bones and weakens bone structure and replaces Zn, blocking oxygen exchange in red blood cells.…”