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DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein.

Duan MR, Nan J, Liang YH, Mao P, Lu L, Li L, Wei C, Lai L, Li Y, Su XD - Nucleic Acids Res. (2007)

Bottom Line: Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs.A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5.These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

View Article: PubMed Central - PubMed

Affiliation: The National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, PR China.

ABSTRACT
WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

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Structure of AtWRKY1-C and multi-sequence alignment. (A) Ribbon representation of AtWRKY1-C domain. The AtWRKY1-C is composed of five β-strands (yellow ribbons), which are numbered from the N-terminus. The zinc ion is shown as a purple sphere and the zinc-coordinating residues are represented by sticks (yellow for C, red for O, blue for N, orange for S). (B) 3D-superimposition of the structure of AtWRKY1-C and the best representative NMR structure of AtWRKY4-C (model 15), using LSQ Fit in O. Macromolecular structures are shown by cartoons, with the AtWRKY1-C colored in yellow and AtWRKY4-C in cyan. The zinc ions are represented as spheres shown by magenta in AtWRKY1-C and orange in AtWRKY4-C. (C) Charge distribution on AtWRKY1-C structure surfaces given by GRASP. Positive charges are represented by blue, negative charges are represented by red. (D) Structure-based sequence alignment of both N-terminus and C-terminus of the nine Arabidopsis WRKY proteins from group I. The zinc-coordinating residues are shown on blue background. Conserved residue elements for stabilizing the structure and recognizing DNA are drawn on red and yellow background, respectively. Residues of β1, highlighted in green but missed in AtWRKY4-C structure, are rather conserved in all C-terminal domains of group I WRKY proteins. Residues marked by stars and triangles are scaffolds of two stable regions. Details of the interactions of the two regions are represented in Figure 3C and D.
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Figure 2: Structure of AtWRKY1-C and multi-sequence alignment. (A) Ribbon representation of AtWRKY1-C domain. The AtWRKY1-C is composed of five β-strands (yellow ribbons), which are numbered from the N-terminus. The zinc ion is shown as a purple sphere and the zinc-coordinating residues are represented by sticks (yellow for C, red for O, blue for N, orange for S). (B) 3D-superimposition of the structure of AtWRKY1-C and the best representative NMR structure of AtWRKY4-C (model 15), using LSQ Fit in O. Macromolecular structures are shown by cartoons, with the AtWRKY1-C colored in yellow and AtWRKY4-C in cyan. The zinc ions are represented as spheres shown by magenta in AtWRKY1-C and orange in AtWRKY4-C. (C) Charge distribution on AtWRKY1-C structure surfaces given by GRASP. Positive charges are represented by blue, negative charges are represented by red. (D) Structure-based sequence alignment of both N-terminus and C-terminus of the nine Arabidopsis WRKY proteins from group I. The zinc-coordinating residues are shown on blue background. Conserved residue elements for stabilizing the structure and recognizing DNA are drawn on red and yellow background, respectively. Residues of β1, highlighted in green but missed in AtWRKY4-C structure, are rather conserved in all C-terminal domains of group I WRKY proteins. Residues marked by stars and triangles are scaffolds of two stable regions. Details of the interactions of the two regions are represented in Figure 3C and D.

Mentions: As transcriptional regulators, WRKY proteins are shown to contain functional nuclear localization signal (NLS) and are targeted to nucleus (27,48). However, the NLS sequences of WRKY proteins are not well conserved, and may be distributed at different locations. The analysis of protein sequences of AtWRKY1 revealed a potential NLS motif KRRKK between residues 273 and 277 near the C-terminal WRKY domain, which is a conserved feature for at least nine members of the group I WRKY proteins in Arabidopsis (Figure 2D). We have thus included this potential NLS motif in the AtWRKY1-C sequence for this study. Introducing the fusion construct of AtWRKY1-C with dimeric GFP (dGFP) into onion epidermal cells, the green fluorescent signal targeted specifically to the nucleus. However, the fluorescence of dGFP alone and fusion protein of AtWRKY1-CΔNLS with dGFP were unevenly distributed throughout the cells (data not shown), which suggested that the potential NLS motif of AtWRKY1-C is indeed responsible for the nuclear localization of AtWRKY1.Figure 2.


DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein.

Duan MR, Nan J, Liang YH, Mao P, Lu L, Li L, Wei C, Lai L, Li Y, Su XD - Nucleic Acids Res. (2007)

Structure of AtWRKY1-C and multi-sequence alignment. (A) Ribbon representation of AtWRKY1-C domain. The AtWRKY1-C is composed of five β-strands (yellow ribbons), which are numbered from the N-terminus. The zinc ion is shown as a purple sphere and the zinc-coordinating residues are represented by sticks (yellow for C, red for O, blue for N, orange for S). (B) 3D-superimposition of the structure of AtWRKY1-C and the best representative NMR structure of AtWRKY4-C (model 15), using LSQ Fit in O. Macromolecular structures are shown by cartoons, with the AtWRKY1-C colored in yellow and AtWRKY4-C in cyan. The zinc ions are represented as spheres shown by magenta in AtWRKY1-C and orange in AtWRKY4-C. (C) Charge distribution on AtWRKY1-C structure surfaces given by GRASP. Positive charges are represented by blue, negative charges are represented by red. (D) Structure-based sequence alignment of both N-terminus and C-terminus of the nine Arabidopsis WRKY proteins from group I. The zinc-coordinating residues are shown on blue background. Conserved residue elements for stabilizing the structure and recognizing DNA are drawn on red and yellow background, respectively. Residues of β1, highlighted in green but missed in AtWRKY4-C structure, are rather conserved in all C-terminal domains of group I WRKY proteins. Residues marked by stars and triangles are scaffolds of two stable regions. Details of the interactions of the two regions are represented in Figure 3C and D.
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Related In: Results  -  Collection

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Figure 2: Structure of AtWRKY1-C and multi-sequence alignment. (A) Ribbon representation of AtWRKY1-C domain. The AtWRKY1-C is composed of five β-strands (yellow ribbons), which are numbered from the N-terminus. The zinc ion is shown as a purple sphere and the zinc-coordinating residues are represented by sticks (yellow for C, red for O, blue for N, orange for S). (B) 3D-superimposition of the structure of AtWRKY1-C and the best representative NMR structure of AtWRKY4-C (model 15), using LSQ Fit in O. Macromolecular structures are shown by cartoons, with the AtWRKY1-C colored in yellow and AtWRKY4-C in cyan. The zinc ions are represented as spheres shown by magenta in AtWRKY1-C and orange in AtWRKY4-C. (C) Charge distribution on AtWRKY1-C structure surfaces given by GRASP. Positive charges are represented by blue, negative charges are represented by red. (D) Structure-based sequence alignment of both N-terminus and C-terminus of the nine Arabidopsis WRKY proteins from group I. The zinc-coordinating residues are shown on blue background. Conserved residue elements for stabilizing the structure and recognizing DNA are drawn on red and yellow background, respectively. Residues of β1, highlighted in green but missed in AtWRKY4-C structure, are rather conserved in all C-terminal domains of group I WRKY proteins. Residues marked by stars and triangles are scaffolds of two stable regions. Details of the interactions of the two regions are represented in Figure 3C and D.
Mentions: As transcriptional regulators, WRKY proteins are shown to contain functional nuclear localization signal (NLS) and are targeted to nucleus (27,48). However, the NLS sequences of WRKY proteins are not well conserved, and may be distributed at different locations. The analysis of protein sequences of AtWRKY1 revealed a potential NLS motif KRRKK between residues 273 and 277 near the C-terminal WRKY domain, which is a conserved feature for at least nine members of the group I WRKY proteins in Arabidopsis (Figure 2D). We have thus included this potential NLS motif in the AtWRKY1-C sequence for this study. Introducing the fusion construct of AtWRKY1-C with dimeric GFP (dGFP) into onion epidermal cells, the green fluorescent signal targeted specifically to the nucleus. However, the fluorescence of dGFP alone and fusion protein of AtWRKY1-CΔNLS with dGFP were unevenly distributed throughout the cells (data not shown), which suggested that the potential NLS motif of AtWRKY1-C is indeed responsible for the nuclear localization of AtWRKY1.Figure 2.

Bottom Line: Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs.A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5.These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

View Article: PubMed Central - PubMed

Affiliation: The National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, PR China.

ABSTRACT
WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

Show MeSH
Related in: MedlinePlus