Abstract
The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys(2)-His(2) motifs, each of which was projected to form well-structured betabetaalpha domains upon Zn(II) binding. In this report, the structure and backbone dynamics of a fragment containing the unusual C-terminal fingers F4-F6 has been investigated. (15)N heteronuclear single quantum coherence (HSQC) spectra of uniformly (15)N-labeled hMTF-zf46 show that Zn(II) induces the folding of hMTF-zf46. Analysis of the secondary structure of Zn(3) hMTF-zf46 determined by (13)Calpha chemical shift indexing and the magnitude of (3)J(Halpha-HN) clearly reveal that zinc fingers F4 and F6 adopt typical betabetaalpha structures. An analysis of the heteronuclear backbone (15)N relaxation dynamics behavior is consistent with this picture and further reveals independent tumbling of the finger domains in solution. Titration of apo-MTF-zf46 with Zn(II) reveals that the F4 domain binds Zn(II) significantly more tightly than do the other two finger domains. In contrast to fingers F4 and F6, the betabetaalpha fold of finger F5 is unstable and only partially populated at substoichiometric Zn(II); a slight molar excess of zinc results in severe conformational exchange broadening of all F5 NH cross-peaks. Finally, although Cd(II) binds to apo-hMTF-zf46 as revealed by intense S(-)-->Cd(II) absorption, a non-native structure results; addition of stoichiometric Zn(II) to the Cd(II) complex results in quantitative refolding of the betabetaalpha structure in F4 and F6. The functional implications of these results are discussed.
Highlights
The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys2-His2 motifs, each of which was projected to form wellstructured ␣ domains upon Zn(II) binding
An analysis of the heteronuclear backbone 15N relaxation dynamics behavior is consistent with this picture and further reveals independent tumbling of the finger domains in solution
An 15N heteronuclear single quantum coherence (HSQC) spectrum is shown in Fig. 3 for a sample of FIG. 3. 15N HSQC spectrum obtained for uniformly 15N-labeled apohMTF-zf46 to which ϳ3 mol eq of Zn(II) was added
Summary
The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys2-His motifs, each of which was projected to form wellstructured ␣ domains upon Zn(II) binding. In many cell types, including mammalian (mouse, rat, and human) cells [11, 12], fish [13], and Drosophila melanogaster [14], zinc-dependent transcriptional regulation of metallothionein (MT) genes plays an important role in zinc homeostasis and detoxification This regulation requires the interaction of metal-response element (MRE)-binding transcription factor-1 (MTF-1) [15] with MREs [16, 17] situated in the promoters of zinc-inducible genes [17]. MTF-1 is a constitutively expressed protein in mouse and human cells of ϳ80 kDa that contains six Cys2His zinc finger domains and multiple domains for transcriptional activation [18], the latter of which likely play important roles in the signal transduction pathway that leads to a zinc response [19]. Recent studies reveal that deletion of F1 from hMTF-zf destroys high affinity MREdbinding activity of the zinc finger domain [36]
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