The conserved basic residues and the charged amino acid residues at the α-helix of the zinc finger motif regulate the nuclear transport activity of triple C2H2 zinc finger proteins

Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins.蛋白质上的锌指（Zinc finger, ZF）基序常被认为是介导DNA结合的经典结构。已有大量研究证实，锌指基序携带有核定位信号（nuclear localization signal, NLS），可介导锌指蛋白向细胞核内转运。本研究聚焦三型C2H2锌指蛋白（triple C2H2 ZF proteins）的核转运调控机制，对其关键影响因子展开探究。在已被报道具有核定位信号功能的三型C2H2锌指蛋白的锌指序列中，我们鉴定出3个保守的碱性残基（热点残基）；此外，锌指的α螺旋上还存在额外的碱性残基。本研究以Egr-1的锌指结构域（Zinc finger domain, ZFD）为模板，构建多种突变体并在细胞中进行表达，通过分析定位于细胞核内的蛋白比例，评估不同突变体的核转运活性。对Egr-1锌指的任意热点残基进行突变，均会降低其核转运活性；将Egr-1锌指结构域第二个锌指（ZF2）的α螺旋区域的碱性残基替换，则会完全消除其核定位信号活性。不过，若将ZF1或ZF3同源位置的酸性残基替换为碱性残基，则可恢复该核定位信号活性；当对ZF1的热点残基或ZF3的α螺旋碱性残基进行突变时，恢复的核转运活性会再次下降。核转运活性的变化与锌指蛋白与输入蛋白（importins）的结合活性直接相关。本研究进一步将探究拓展至其他三型C2H2锌指蛋白：与Egr-1类似，SP1与KLF家族的锌指结构域在α螺旋的第二位（α2）与第三位（α3）位点带有带电氨基酸残基；将α2与α3位点的氨基酸替换为酸性残基，会降低SP1与KLF6锌指结构域的核定位信号活性；若将任意SP1与KLF6锌指结构域的α3位点替换为组氨酸，则可恢复其受损的核定位信号活性。上述结果再次证实，锌指结构与α螺旋上的带电残基在调控三型C2H2锌指蛋白的核定位信号活性中存在可互换的作用。