Structural and functional heterogeneity among the zinc fingers of human MRE-binding transcription factor-1

MRE-binding transcription factor-1 (MTF-1) activates the expression of metallothionein (MT) genes in mouse and human cells upon binding to one or more tandem metal-response elements (MREs; 5'-ctnTGCRCnCgGCCc) in the MT promoter. MTF-1 contains six Cys(2)-His(2) zinc finger sequences. Previous work suggests that the zinc finger domain itself may function as a zinc sensor in zinc-activated expression of MTs. To obtain molecular insight into MTF-1 function, a recombinant fragment of MTF-1 containing only the zinc finger domain (denoted MTF-zf) has been purified using nondenaturing conditions and characterized with respect to zinc-binding properties, secondary structure, and DNA-binding activity. Different preparations of MTF-zf, following an anaerobic dialysis to quantify Zn(II) and reduced cysteine (by DTNB reactivity) content, reveal Zn(II)/MTF-zf stoichiometries ranging from 3.3 to 5.5 g at Zn(II) and 11-13 reduced thiolates (12 expected). Far-UV CD spectra reveal indistinguishable secondary structural content in all preparations, i.e., enough to fold just three of six zinc fingers of MTF-zf. Removal of additional zinc from MTF-zf gives rise to an insoluble apoprotein. Complex formation between a Zn-5.5 MTF-zf and a coumarin-labeled MREd-containing oligonucleotide as monitored by changes in the anisotropy of the coumarin fluorescence gives a K-app = 3.8 (+/-0.5) x 10(8) M-1 (pH 7.0, 0.20 M NaCl, 25 degrees C). Investigation of the salt type and concentration dependence of K-app suggests significant contributions from both cation and anion release upon complex formation. Zn-5.5 MTF-zf exhibits a large negative heat capacity of complex formation with MREd and can discriminate among DNA duplexes which have mutations deposited on either the TGCRC core or the C-rich side of the MREd. Air oxidation of Zn-5.5 MTF-zf results in the reversible conversion of 6 of the 12 Cys thiolates to 3 disulfide bonds; as expected, this has no effect on the secondary structure of MTF-zf, but results in approximate to 30-fold reduction in K-app to approximate to 1.2 x 10(7) M-1. In contrast, fully reduced Zn-3.5 MTF-zf binds to the MREd with an affinity and [NaCl] dependence largely indistinguishable from those of Zn-5.5 MTF-zf. The zinc fingers in MTF-zf are physically and functionally inequivalent, A subset (approximate to 3-4) of zinc fingers plays a structural role in folding and high-affinity MREd binding, while one or more additional fingers have properties potentially consistent with a metalloregulatory role.