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.

Chih-Ying Lin,Lih-Yuan Lin,Mark Isalan

doi:10.1371/journal.pone.0191971

Abstract

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.

Highlights

Zinc finger domain (ZFD) is among the most common DNA binding structures found in eukaryotic transcription factors
The ZFD sequence of several triple C2H2 ZF proteins (Egr-1 [38], KLF1 [44], KLF6 [41], KLF8 [50], and SP1 [46]) that reportedly act as nuclear localization signal (NLS) were aligned
The proportion of cells having the green fluorescent proteins (GFP) fusion protein entirely localized in the nucleus was determined

Summary

Introduction

Zinc finger domain (ZFD) is among the most common DNA binding structures found in eukaryotic transcription factors. 700 genes encoding C2H2 ZFD have been identified in the human genome. They represent the most abundant DNA binding proteins in cells [4]. C2H2 ZFD was identified initially in the transcription factor TFIIIA of Xenopus laevis [5] and presumed to present only in eukaryotic cells. Later studies indicate that proteins with C2H2 ZFD exist in prokaryotic cells, suggesting a structural conservation in evolution [6]. Besides DNA binding, the C2H2 motif of the ZF proteins participates in RNA binding [7, 8] and protein-protein interactions [9]

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Results

Conclusion