MRE-binding transcription factor-1: Weak zinc-binding finger domains 5 and6 modulate the structure, affinity, and specificity of the metal-response element complex

MRE-binding transcription factor-1 (MTF-1) contains six Cys(2)-His(2) zinc finger sequences, and it has been suggested that the zinc finger domain its elf may function as a zinc sensor in zinc-activated expression of metalloth ioneins (MTs). Previous work has shown that a subset (approximate to 3-4) o f the zinc fingers in MTF-zf play a structural role in folding and high-aff inity metal-response element (MREd) binding, while one or more other finger s have properties consistent with a metalloregulatory role (weak zinc bindi ng affinity in the absence of DNA). We show here that zinc fingers 5 and 6 correspond to the weak zinc-binding fingers in MTF-zf. Limited trypsinolysi s of a Zn-6-MTF-zf:MREd complex gives rise to a highly protease-resistant c ore fragment corresponding to amino acids 137-260 or N-terminal zinc finger s 1-4 of MTF-zf. Characterization of a collection of broken-finger (His --> Asn) and missing-finger mutants of MTF-zf reveals that deletion of zinc fi ngers 5 and 6 to create MTF-zf14 attenuates MREd binding affinity (approxim ate to 20-fold), while deletion of fingers 4-6 (MTF-zf13) results in a furt her 20-fold reduction of binding affinity with a nearly complete loss of sp ecificity. Circular dichroism studies reveal that the binding of MTF-zf to the MREd induces a dramatic alteration of the structure of the MREd from a B-form to a double-helical conformation with A-like features. Formation of stoichiometric complexes with MTF-2f14, H279N (Delta zf5) MTF-zf, and MTF-z f13 induces comparatively less A-like structure. Steady-state fluorescence resonance energy transfer (FRET) spectroscopy has been used to globally def ine the orientation of the multifinger MTF-zf on the MREd. These experiment s suggest that fingers 1-4 are oriented on the highly conserved TGCRCnC sid e of the MREd with fingers 5-6 bound at or near the gGCCc sequence. These f indings are consistent with a model in which the N-terminal zinc fingers in MTF-zf are required for high affinity and specific binding to the consensu s TGCRCnC core in a way which is subjected to structural and allosteric mod ulation by the weak zinc-binding C-terminal zinc fingers.