Conformational Heterogeneity in the C-terminal Zinc Fingers of Human MTF-1. An NMR and zinc-binding study

Abstract

The human metalloregulatory transcription factor, metal-response element (MRE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys 2-His2 motifs, each of which was projected to form well-structured ββα 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. 15N heteronuclear single quantum coherence (HSQC) spectra of uniformly 15N-labeled hMTF-zf46 show that Zn(II) induces the folding of hMTF-zf46. Analysis of the secondary structure of Zn3 hMTF-zf46 determined by 13Cα chemical shift indexing and the magnitude of 3JHα-HN clearly reveal that zinc fingers F4 and F6 adopt typical ββα structures. 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. 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 ββα 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 ββα structure in F4 and F6. The functional implications of these results are discussed.