Conformational heterogeneity in the C-terminal zinc fingers of human MTF-1: an NMR and zinc-binding study.
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.
Giedroc
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DP
Department of Biochemistry and Biophysics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-2128, USA. giedroc@tamu.edu.
Chen
X
X
Pennella
M A
MA
LiWang
A C
AC
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011487:Protein Conformation; D014157:Transcription Factors / Q000737:chemistry*; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers; D000097809:Transcription Factor MTF-1
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1