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Article

Spectroscopic and Functional Determination of the Interaction of Pb²⁺ with GATA Proteins

Supporting Info

Amy B. Ghering,^† Lisa M. Miller Jenkins,^‡ Brandy L. Schenck,^§ Sandhya Deo,^† R. Aeryn Mayer,^† Michael J. Pikaart,^§ James G. Omichinski, and Hilary A. Godwin^†*

Contribution from the Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, Department of Chemistry and Biochemistry, Hope College, 35 East 12th Street, Holland, Michigan 49423, Département de Biochimie, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, Qc. Canada, H3C 3J7, and Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 North Campus Drive, Evanston, Illinois 60208-3300

J. Am. Chem. Soc., 2005, 127 (11), pp 3751–3759

DOI: 10.1021/ja0464544

Publication Date (Web): February 23, 2005

†

Department of Chemistry, Northwestern University.

‡

University of Georgia.

Hope College.

‖

Université de Montréal.

⊥

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University.

In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

h-godwin@northwestern.edu

Abstract

GATA proteins are transcription factors that bind GATA DNA elements through Cys₄ structural zinc-binding domains and play critical regulatory roles in neurological and urogenital development and the development of cardiac disease. To evaluate GATA proteins as potential targets for lead, spectroscopically monitored metal-binding titrations were used to measure the affinity of Pb²⁺ for the C-terminal zinc-binding domain from chicken GATA-1 (CF) and the double-finger domain from human GATA-1 (DF). Using this method, Pb²⁺ coordinating to CF and DF was directly observed through the appearance of intense bands in the near-ultraviolet region of the spectrum (250−380 nm). Absorption data collected from these experiments were best fit to a 1:1 Pb²⁺−CF model and a 2:1 Pb²⁺−DF model. Competition experiments using Zn²⁺ were used to determine the absolute affinities of Pb²⁺ for these proteins. These studies reveal that Pb²⁺ forms tight complexes with cysteine residues in the zinc-binding sites in GATA proteins, β₁^Pb = 6.4 (±2.0) × 10⁹ M^-¹ for CF and β₂ = 6.3 (±6.3) × 10¹⁹ M^-² for Pb²⁺₂−DF, and within an order of magnitude of the affinity of Zn²⁺ for these proteins. Furthermore, Pb²⁺ was able to displace bound Zn²⁺ from CF and DF. Upon addition of Pb²⁺, GATA shows a decreased ability to bind to DNA and subsequently activate transcription. Therefore, the DNA binding and transcriptional activity of GATA proteins are most likely to be targeted by Pb²⁺ in cells and tissues that sequester Pb²⁺ in vivo, which include the brain and the heart.