Domain Z7 of nuclear transcription factor ZNF711 has the consensus last metal-ligand H23 found in odd-numbered zinc-fingers of this protein replaced by a phenylalanine. Ever since the discovery of ZNF711 it has been thought that Z7 is probably non-functional because of the H23F substitution. The presence of H26 three positions downstream prompted us to examine if this histidine could substitute as the last metal ligand. The Z7 domain adopts a stable tertiary structure upon metal binding. The NMR structure of Zn²⁺-bound Z7 shows the classical ββα-fold of CCHH zinc fingers. Mutagenesis and pH titration experiments indicate that H26 is not involved in metal binding and that Z7 has a tridentate metal-binding site comprised of only residues C3, C6, and H19. By contrast, an F23H mutation that introduces a histidine in the consensus position forms a tetradentate ligand. The structure of the WT Z7 is stable causing restricted ring-flipping of phenyalanines 10 and 23. Dynamics are increased with either the H26A or F23H substitutions and aromatic ring rotation is no longer hindered in the two mutants. The mutations have only small effects on the K_d values for Zn²⁺ and Co²⁺ and retain the high thermal stability of the WT domain above 80 °C. Like two previously reported designed zinc fingers with the last ligand replaced by water, the WT Z7 domain is catalytically active, hydrolyzing 4-nitophenyl acetate. We discuss the implications of naturally occurring tridentate zinc fingers for cancer mutations and drug targeting of notoriously undruggable transcription factors. Our findings that Z7 can fold with only a subset of three metal ligands suggests the recent view that most everything about protein structure can be predicted through homology modeling might be premature for at least the resilient and versatile zinc-finger motif.

中文翻译：

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通过实验 NMR 结构修复转录因子 ZNF711 先前简并的第七锌指结构域

核转录因子 ZNF711 的结构域 Z7 具有在该蛋白质的奇数锌指中发现的最后一个共有金属配体 H23，该配体被苯丙氨酸取代。自从发现 ZNF711 以来，人们一直认为 Z7 可能由于 H23F 取代而失去功能。 H26 下游三个位置的存在促使我们检查该组氨酸是否可以替代作为最后一个金属配体。 Z7 结构域在金属结合后采用稳定的三级结构。 Zn ²⁺结合的Z7的NMR 结构显示了CCHH 锌指的经典ββα 折叠。诱变和 pH 滴定实验表明，H26 不参与金属结合，而 Z7 具有仅由残基 C3、C6 和 H19 组成的三齿金属结合位点。相比之下，在共有位置引入组氨酸的 F23H 突变形成四齿配体。 WT Z7 的结构是稳定的，导致苯丙氨酸 10 和 23 的环翻转受到限制。H26A 或 F23H 取代可增加动力学，并且两个突变体中芳环旋转不再受到阻碍。这些突变对 Zn ²⁺和 Co ^2+的K _d值影响很小，并且保留了 WT 结构域在 80 °C 以上的高热稳定性。与之前报道的两个设计的锌指（最后一个配体被水取代）一样，WT Z7 结构域具有催化活性，可水解 4-硝基苯乙酸酯。我们讨论了天然存在的三齿锌指对癌症突变和众所周知的不可成药转录因子的药物靶向的影响。我们发现 Z7 只能与三种金属配体的子集折叠，这表明最近关于蛋白质结构的大部分内容都可以通过同源模型预测的观点可能还为时过早，至少对于弹性和多功能的锌指基序而言。