The Six Zinc Fingers of Metal-responsive Element Binding Transcription Factor-1 Form Stable and Quasi-ordered Structures with Relatively Small Differences in Zinc Affinities

Six Cys2His2 zinc fingers (F1–6) comprise the DNA binding domain of metal-responsive element binding transcription factor-1 (MTF-1). F1–6 is necessary for basal and zinc-induced expression of metallothionein genes. Analysis of NMR structural and dynamic data for an F1–6 protein construct demonstrates that each zinc finger adopts a stable ββα fold in the presence of stoichiometric Zn(II), provided that all cysteine ligands are in a reduced state. Parallel studies of protein constructs spanning the four N-terminal core DNA binding fingers (F1–4) and two C-terminal low DNA affinity fingers (F5–6) reveal similar stable zinc finger structures. In both the F1–6 and F5–6 proteins, the finger 5 cysteines were found to readily oxidize at neutral pH. Detailed spectral density and hydrodynamic analysis of 15N relaxation data revealed quasi-ordered anisotropic rotational diffusion properties of the six F1–6 zinc fingers that could influence MTF-1 DNA binding function. A more general effect on the rotational diffusion properties of Cys2His2 zinc fingers was also uncovered that is dependent upon the position of each finger within multifinger domains. Analysis of NMR 1H-15N-heteronuclear single quantum coherence spectral peak intensities measured as a function of added Zn(II) in conjunction with Zn(II) binding modeling studies indicated that the Zn(II) affinities of all MTF-1 zinc fingers are within ∼10–50-fold. These analyses further suggested that metal sensing by MTF-1 in eukaryotic cells involves multiple zinc fingers and occurs over a 100-fold or less range of accessible Zn(II) concentration. Six Cys2His2 zinc fingers (F1–6) comprise the DNA binding domain of metal-responsive element binding transcription factor-1 (MTF-1). F1–6 is necessary for basal and zinc-induced expression of metallothionein genes. Analysis of NMR structural and dynamic data for an F1–6 protein construct demonstrates that each zinc finger adopts a stable ββα fold in the presence of stoichiometric Zn(II), provided that all cysteine ligands are in a reduced state. Parallel studies of protein constructs spanning the four N-terminal core DNA binding fingers (F1–4) and two C-terminal low DNA affinity fingers (F5–6) reveal similar stable zinc finger structures. In both the F1–6 and F5–6 proteins, the finger 5 cysteines were found to readily oxidize at neutral pH. Detailed spectral density and hydrodynamic analysis of 15N relaxation data revealed quasi-ordered anisotropic rotational diffusion properties of the six F1–6 zinc fingers that could influence MTF-1 DNA binding function. A more general effect on the rotational diffusion properties of Cys2His2 zinc fingers was also uncovered that is dependent upon the position of each finger within multifinger domains. Analysis of NMR 1H-15N-heteronuclear single quantum coherence spectral peak intensities measured as a function of added Zn(II) in conjunction with Zn(II) binding modeling studies indicated that the Zn(II) affinities of all MTF-1 zinc fingers are within ∼10–50-fold. These analyses further suggested that metal sensing by MTF-1 in eukaryotic cells involves multiple zinc fingers and occurs over a 100-fold or less range of accessible Zn(II) concentration. Organisms ranging from bacteria to mammals maintain intracellular Zn(II) levels within a functional range through homeostatic mechanisms that include metal-dependent regulation of gene expression. It has recently been proposed that the high Zn(II) binding affinities reported for the bacterial Zur and ZntR proteins suggest that Zn(II) homeostasis in Escherichia coli is tightly regulated, such that essentially no labile or “accessible” cytoplasmic Zn(II) is available (1.Outten C.E. O'Halloran T.V. Science. 2001; 292: 2488-2492Crossref PubMed Scopus (1173) Google Scholar). In this model, Zn(II) substitution reactions between Zur and ZntR and as yet unidentified metal chaperone transporters could mediate the metal response. In higher eukaryotic organisms, multiple homeostatic mechanisms regulate intracellular Zn(II) levels (2.Andrews G.K. Biometals. 2001; 14: 223-237Crossref PubMed Scopus (284) Google Scholar, 3.Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (433) Google Scholar). However, the accessible range over Zn(II) is in eukaryotic cells is In higher eukaryotic organisms, expression of for the zinc and metallothionein of proteins is through the metal-responsive element binding transcription factor-1 metal-responsive element binding transcription DNA binding domain of MTF-1 six Cys2His2 zinc four N-terminal zinc fingers of two C-terminal zinc fingers of metal-responsive DNA single quantum relaxation relaxation rotational the N-terminal zinc fingers of from the four zinc fingers from the in are the of MTF-1 and are for Zn(II) and metal MTF-1 transcription was from PubMed Scopus Google and has been in PubMed Scopus Google Scholar, PubMed Google 2001; PubMed Scopus Google Google PubMed Scopus Google Scholar, PubMed Scopus Google and and MTF-1 is a protein that a zinc finger DNA binding domain (F1–6) PubMed Scopus Google and the of the between all of MTF-1 in the zinc finger domain Google Scholar). and MTF-1 zinc finger by a single occurs in the of high of the MTF-1 DNA binding domain is with a proposed Zn(II) of MTF-1 is through a metal-responsive DNA element is on the of a that a on the PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). in are found in multiple within the of genes. of to zinc finger metal as an to of on from higher eukaryotic that MTF-1 binding to the is by the presence of Zn(II) at PubMed Google Scholar, G.K. 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