title: Spectroscopic and functional determination of the interaction of Pb2+ with GATA proteins.
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, beta1Pb = 6.4 (+/- 2.0) x 10(9) M(-1) for CF and beta2 = 6.3 (+/- 6.3) x 10(19) M(-2) for Pb(2+)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.
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, USA.
authors.forename: Amy B
authors.initials: AB
authors.lastname: Ghering
authors.forename: Lisa M Miller
authors.initials: LM
authors.lastname: Jenkins
authors.forename: Brandy L
authors.initials: BL
authors.lastname: Schenck
authors.forename: Sandhya
authors.initials: S
authors.lastname: Deo
authors.forename: R Aeryn
authors.initials: RA
authors.lastname: Mayer
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pikaart
authors.forename: James G
authors.initials: JG
authors.lastname: Omichinski
authors.forename: Hilary A
authors.initials: HA
authors.lastname: Godwin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002413:Cations, Divalent; D002645:Chickens; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006801:Humans; D007700:Kinetics; D007854:Lead / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D017186:Titrimetry; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D015032:Zinc / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors; D007854:Lead; D004247:DNA; D015032:Zinc