NMR identification of heavy metal-binding sites in a synthetic zinc finger peptide: toxicological implications for the interactions of xenobiotic metals with zinc finger proteins.
Lead (Pb), mercury (Hg), and cadmium (Cd) are toxic and interfere with protein metal-binding sites. The Cys(2)/His(2) zinc finger is a structural motif required for sequence-specific DNA binding and is present in zinc finger transcription factors (ZFP): Sp1, Egr-1, and TFIIIA. Neurotoxic studies have shown that heavy metals directly inhibit the DNA binding of ZFP and result in adverse cellular effects. Recently, we demonstrated the ability of heavy metals to alter the DNA binding of a synthetic Cys(2)/His(2) finger peptide (Razmiafshari and Zawia, Toxicol. Appl. Pharmacol. 166, 1-12, 2000). To determine the precise site of interactions between heavy metals and this protein domain, Pb, Hg, Cd, and Ca were reconstituted with the synthetic apopeptide and studied by one- and two-dimensional NMR spectroscopy. In the presence of Zn, Cd, Hg, and Pb, but not Ca, distinct peptide NMR signal changes in the aliphatic region were observed and attributed to metal-cystiene interactions. 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 presence and absence of Zn, Pb, and Ca through TOCSY and NOESY spectra. Cysteine and histidine residues showed a distinct change in their amide and beta resonances 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 the binding site. These studies reveal this structure as a mediator of metal-induced alterations in protein function.
Community and Environmental Medicine, University of California, Irvine, California, 92697-1820, USA.
M
M
Razmiafshari
J
J
Kao
A
A
d'Avignon
N H
NH
Zawia
D001667:Binding, Competitive / Q000187:drug effects; D004247:DNA / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D019216:Metals, Heavy / Q000378:metabolism* / Q000633:toxicity; D010455:Peptides / Q000187:drug effects / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D015262:Xenobiotics / Q000378:metabolism* / Q000633:toxicity; D016335:Zinc Fingers / Q000187:drug effects* / Q000502:physiology
D004268:DNA-Binding Proteins; D019216:Metals, Heavy; D010455:Peptides; D015262:Xenobiotics; D004247:DNA