Chen X, Chu M, Giedroc D P
Department of Biochemistry and Biophysics, Center for Macromolecular Design, Texas A&M University, College Station, Texas 77843-2128, USA.
Biochemistry. 1999 Sep 28;38(39):12915-25. doi: 10.1021/bi9913000.
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.
MRE结合转录因子1（MTF-1）含有六个Cys(2)-His(2)锌指序列，有人提出锌指结构域本身可能在金属硫蛋白（MTs）的锌激活表达中作为锌传感器发挥作用。先前的研究表明，MTF-zf中的一部分锌指（约3 - 4个）在折叠和高亲和力金属反应元件（MREd）结合中起结构作用，而一个或多个其他锌指具有与金属调节作用一致的特性（在无DNA时锌结合亲和力弱）。我们在此表明，锌指5和6对应于MTF-zf中弱锌结合锌指。对Zn(6)-MTF-zf:MREd复合物进行有限的胰蛋白酶消化会产生一个高度抗蛋白酶的核心片段，对应于MTF-zf的氨基酸137 - 260或N端锌指1 - 4。对MTF-zf的一系列断指（His→Asn）和缺指突变体的表征表明，缺失锌指5和6以创建MTF-zf14会减弱MREd结合亲和力（约20倍），而缺失指4 - 6（MTF-zf13）会导致结合亲和力进一步降低20倍，且特异性几乎完全丧失。圆二色性研究表明，MTF-zf与MREd的结合会使MREd的结构从B型显著改变为具有A样特征的双螺旋构象。与MTF-zf14、H279N（Deltazf5）MTF-zf和MTF-zf13形成化学计量复合物会诱导相对较少的A样结构。稳态荧光共振能量转移（FRET）光谱已用于全面确定多锌指MTF-zf在MREd上的取向。这些实验表明，指1 - 4定位于MREd高度保守的TGCRCnC一侧，指5 - 6结合在gGCCc序列处或附近。这些发现与一个模型一致，在该模型中，MTF-zf中的N端锌指对于以一种受弱锌结合C端锌指的结构和变构调节的方式与共有TGCRCnC核心进行高亲和力和特异性结合是必需的。
