Spectroscopic determination of the thermodynamics of cobalt and zinc binding to GATA proteins.
Vertebrate GATA proteins regulate processes that are vital to development, and each possesses two tandem GATA finger domains: an N-terminal GATA finger and a C-terminal GATA finger. These GATA fingers require Zn(2+) to fold, to bind DNA recognition elements, and to regulate transcription. While the GATA-1 C-terminal finger is necessary and sufficient to bind to single GATA DNA sites, the N-terminal finger interacts with DNA such that the double finger unit (DF domain) has a binding and transactivation profile that is tuned by the DNA-binding site. Co(2+) was used as a spectroscopic probe in a series of competition titrations to determine the affinity of Co(2+) and Zn(2+) for the C-terminal finger from chicken GATA-1 and the double finger from human GATA-1 (referred to in this report as CF and DF). For CF, these experiments yielded K(b)(Co) = 1.0 (+/-1.3) x 10(7) M(-1) and K(b)(Zn) = 2.0 (+/-1.3) x 10(10) M(-1). For DF, these experiments yielded equilibrium constants for the process of two M(2+) binding to form M(2+)(2)-DF of beta(2)(Co) = 2.5 (+/-1.6) x 10(14) M(-2) and beta(2)(Zn) = 6.3 (+/-2.5) x 10(20) M(-2). The ZnS(4) coordination environment of Zn(2+)-bound CF was confirmed with X-ray absorption spectroscopy. A detailed analysis of these data suggests that the N-terminal and C-terminal fingers of DF act as independent and identical Zn(2+)-binding sites and each finger binds Zn(2+) with an affinity equivalent to that of CF.
Ghering
Amy B
AB
Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, USA.
Shokes
Jacob E
JE
Scott
Robert A
RA
Omichinski
James G
JG
Godwin
Hilary A
HA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry / Q000379:methods*; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry*; D014158:Transcription, Genetic; D015032:Zinc / Q000737:chemistry*
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D007477:Ions; D008670:Metals; D014157:Transcription Factors; D003035:Cobalt; D004247:DNA; D015032:Zinc