MRE-Binding transcription factor-1: weak zinc-binding finger domains 5 and 6 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 itself may function as a zinc sensor in zinc-activated expression of metallothioneins (MTs). Previous work has shown that a subset ( approximately 3-4) of the zinc fingers in MTF-zf play a structural role in folding and high-affinity metal-response element (MREd) binding, while one or more other fingers have properties consistent with a metalloregulatory role (weak zinc binding 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 trypsinolysis of a Zn(6)-MTF-zf:MREd complex gives rise to a highly protease-resistant core fragment corresponding to amino acids 137-260 or N-terminal zinc fingers 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 fingers 5 and 6 to create MTF-zf14 attenuates MREd binding affinity ( approximately 20-fold), while deletion of fingers 4-6 (MTF-zf13) results in a further 20-fold reduction of binding affinity with a nearly complete loss of specificity. 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-zf14, H279N (Deltazf5) MTF-zf, and MTF-zf13 induces comparatively less A-like structure. Steady-state fluorescence resonance energy transfer (FRET) spectroscopy has been used to globally define the orientation of the multifinger MTF-zf on the MREd. These experiments suggest that fingers 1-4 are oriented on the highly conserved TGCRCnC side of the MREd with fingers 5-6 bound at or near the gGCCc sequence. These findings 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 consensus TGCRCnC core in a way which is subjected to structural and allosteric modulation by the weak zinc-binding C-terminal zinc fingers.
Chen
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Department of Biochemistry and Biophysics, Center for Macromolecular Design, Texas A&M University, College Station, Texas 77843-2128, USA.
Chu
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Giedroc
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA; D004268:DNA-Binding Proteins; D004735:Energy Transfer; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D016335:Zinc Fingers; D000097809:Transcription Factor MTF-1
D004247:DNA; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D014357:Trypsin; D000097809:Transcription Factor MTF-1