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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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CD spectra. CD spectra of wild-type AtWRKY1-C and the mutant proteins on β2 strand. The different proteins were distinguished from each other by colors. (A) CD spectra of wild-type AtWRKY1-C and the mutant proteins on β3 and β4 strands.
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Figure 6: CD spectra. CD spectra of wild-type AtWRKY1-C and the mutant proteins on β2 strand. The different proteins were distinguished from each other by colors. (A) CD spectra of wild-type AtWRKY1-C and the mutant proteins on β3 and β4 strands.

Mentions: After searching for similar structures of AtWRKY1-C using the DALI server (52), a DNA–protein complex structure, the Drosophila GCM (44) (PDB ID 1ODH, Z-score > 6.2) was returned. When the GCM protein was used to perform a least-square fitting with AtWRKY1-C, the resulting RMSD between the aligned parts (68 residues) was 2.3 Å, a DNA complex model of W-box DNA and AtWRKY1-C was thus constructed as shown in Figure 7A. By comparison of the potential DNA-binding residues (which are close to the DNA major groove as shown in the complex model), and previous work on the DNA-binding related residues on the WRKYGQK motif (29), the following mutants with a single residue change were constructed: R313E, K314A, K314R, Y315F, Y315R, G316F, Q317A, Q317K, R327A, R327E, Y330F, R331K, R331A and K340A. In Maeo et al.'s work (29), it has been shown that all residues in the WRKYGQK (corresponding to residues 312–318 in AtWRKY1-C) motif are important for the DNA binding. Particularly for W312, K314, Y315, K318, a change to Ala for any of those residues would completely abolish the DNA binding. On the other hand, the R313, G316 and Q317 would only reduce the amount of DNA binding (29). We have thus constructed multiple mutants for R313, K314, Y315, G316 and Q317. Furthermore, the well-conserved residues R327, Y330, R331 and K340 are quite close to the DNA-binding sites, these residues were also selected for mutagenesis studies. Thirteen out of the fourteen mutants are located on strands β2 and β3. This region, particularly the connecting loop, showed flexible feature (high B-factor) in Figure 3A and B, indicating conformational changes might occur upon DNA binding. EMSA were used to test the DNA-binding abilities of these mutants with a fragment of 17-bp duplex DNA containing the W-box of TGAC as shown in Figure 5. The EMSA assay using 32P-labeled probe according to standard protocol was also performed with similar results (not shown). To examine the specificity of the binding, a competition experiment was performed by using an excess amount of unlabeled probe of the same sequence. When a 100-fold excess of unlabeled oligonucleotides were included in the reaction, binding of AtWRKY1-C to the labeled probe was abolished completely (data not shown). All the mutants could be readily expressed and purified as the wild-type AtWRKY1-C, the circular dichroism (CD) spectra of these mutant proteins were used to monitor the correct folding of these proteins, thus the DNA-binding ability changes are not due to the structural change caused by the mutations, see Figure 6.Figure 5.


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)

CD spectra. CD spectra of wild-type AtWRKY1-C and the mutant proteins on β2 strand. The different proteins were distinguished from each other by colors. (A) CD spectra of wild-type AtWRKY1-C and the mutant proteins on β3 and β4 strands.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
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Figure 6: CD spectra. CD spectra of wild-type AtWRKY1-C and the mutant proteins on β2 strand. The different proteins were distinguished from each other by colors. (A) CD spectra of wild-type AtWRKY1-C and the mutant proteins on β3 and β4 strands.
Mentions: After searching for similar structures of AtWRKY1-C using the DALI server (52), a DNA–protein complex structure, the Drosophila GCM (44) (PDB ID 1ODH, Z-score > 6.2) was returned. When the GCM protein was used to perform a least-square fitting with AtWRKY1-C, the resulting RMSD between the aligned parts (68 residues) was 2.3 Å, a DNA complex model of W-box DNA and AtWRKY1-C was thus constructed as shown in Figure 7A. By comparison of the potential DNA-binding residues (which are close to the DNA major groove as shown in the complex model), and previous work on the DNA-binding related residues on the WRKYGQK motif (29), the following mutants with a single residue change were constructed: R313E, K314A, K314R, Y315F, Y315R, G316F, Q317A, Q317K, R327A, R327E, Y330F, R331K, R331A and K340A. In Maeo et al.'s work (29), it has been shown that all residues in the WRKYGQK (corresponding to residues 312–318 in AtWRKY1-C) motif are important for the DNA binding. Particularly for W312, K314, Y315, K318, a change to Ala for any of those residues would completely abolish the DNA binding. On the other hand, the R313, G316 and Q317 would only reduce the amount of DNA binding (29). We have thus constructed multiple mutants for R313, K314, Y315, G316 and Q317. Furthermore, the well-conserved residues R327, Y330, R331 and K340 are quite close to the DNA-binding sites, these residues were also selected for mutagenesis studies. Thirteen out of the fourteen mutants are located on strands β2 and β3. This region, particularly the connecting loop, showed flexible feature (high B-factor) in Figure 3A and B, indicating conformational changes might occur upon DNA binding. EMSA were used to test the DNA-binding abilities of these mutants with a fragment of 17-bp duplex DNA containing the W-box of TGAC as shown in Figure 5. The EMSA assay using 32P-labeled probe according to standard protocol was also performed with similar results (not shown). To examine the specificity of the binding, a competition experiment was performed by using an excess amount of unlabeled probe of the same sequence. When a 100-fold excess of unlabeled oligonucleotides were included in the reaction, binding of AtWRKY1-C to the labeled probe was abolished completely (data not shown). All the mutants could be readily expressed and purified as the wild-type AtWRKY1-C, the circular dichroism (CD) spectra of these mutant proteins were used to monitor the correct folding of these proteins, thus the DNA-binding ability changes are not due to the structural change caused by the mutations, see Figure 6.Figure 5.

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