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 (M RE)-binding transcription factor-1 (MTF-1), contains six TFIIIA-type Cys(2) -His(2) motifs, each of which was projected to form well-structured beta be ta alpha domains upon Zn(II) binding. In this report, the structure and bac kbone dynamics of a fragment containing the unusual C-terminal fingers F4-F 6 has been investigated. N-15 heteronuclear single quantum coherence (HSQC) spectra of uniformly N-15-labeled hMTF-zf46 show that Zn(ll) induces the f olding of hMTF-zf46. Analysis of the secondary structure of Zn-3 hMTF-zf46 determined by C-13 alpha chemical shift indexing and the magnitude of (3)J( H alpha -HN) clearly reveal that zinc fingers F4 and F6 adopt typical beta beta alpha structures. An analysis of the heteronuclear backbone N-15 relax ation dynamics behavior is consistent with this picture and further reveals independent tumbling of the finger domains in solution. Titration of apo-M TF-zf46 with Zn(H) reveals that the F4 domain binds Zn(II) significantly mo re tightly than do the other two finger domains. In contrast to fingers F4 and F6, the beta beta alpha fold of finger F5 is unstable and only partiall y populated at substoichiometric Zn(II); a slight molar excess of zinc resu lts 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 stoichiome tric Zn(II) to the Cd(Il) complex results in quantitative refolding of the beta beta alpha structure in F4 and F6. The functional implications of thes e results are discussed.