NMR identification of heavy metal-binding sites in a synthetic zinc fingerpeptide: Toxicological implications for the interactions of xenobiotic metals with zinc finger proteins

Lead (Pb), mercury (Hg), and cadmium (Cd) are toxic and interfere with prot ein metal-binding sites. The Cys(2)/His(2) zinc finger is a structural moti f required for sequence-specific DNA binding and is present in zinc finger transcription factors (ZFP): Spl, Egr-l, and TFIIIA. Neurotoxic studies hav e shown that heavy metals directly inhibit the DNA binding of ZFP and resul t in adverse cellular effects. Recently, we demonstrated the ability of hea vy metals to alter the DNA binding of a synthetic Cys(2)/His(2) finger pept ide (Razmiafshari and Zawia, Toxicol. Appl. Pharmacol. 166, 1-12, 2000). To determine the precise site of interactions between heavy metals and this p rotein domain, Pb, Hg, Cd, and Ca were reconstituted with the synthetic apo peptide and studied by one- and two-dimensional NMR spectroscopy. In the pr esence of Zn, Cd, Hg, and Pb, but not Ca, distinct peptide NMR signal chang es in the aliphatic region were observed and attributed to metal-cystiene i nteractions. However, chemical shifts indicative of metal-histidine binding were elicited by all the metals in the peptide's aromatic region. Chemical shift assignments and sequential connectivity were established in the pres ence and absence of Zn, Pb, and Ca through TOCSY and NOESY spectra. Cystein e and histidine residues showed a distinct change in their amide and beta r esonances in the presence of Zn and Pb, suggesting the metal-ligand binding sites were near these residues. However, Ca led to no significant spectral changes in these regions, suggesting that it is not actively involved in t he binding site. These studies reveal this structure as a mediator of metal -induced alterations in protein function. (C) 2001 Academic Press