1152 Nucleic Acids Research, 2007, Vol. 35, No. 4 Figure 6. CD spectra. CD spectra of wild-type AtWRKY1-C and the mutant proteins on b2 strand. The diﬀerent proteins were distinguished from each other by colors. (A) CD spectra of wild-type AtWRKY1-C and the mutant proteins on b3 and b4 strands. involved in speciﬁc recognition, and the large-sized residues (not only charged), such as lysines at i and i þ 4, as well as arginines at j and j þ 4, could bridge the large gap separating the DNA. The structure model of DNA–AtWRKY1-C complex we made in this study has been based on the GCM–DNA structure (44), which is diﬀerent from the Tateno’s DNA- binding model (53) in details. The b-strands of the GCM protein adapted a previously undescribed binding mode to interact with DNA (44). Despite of the absence of sequence and topology similarity, AtWRKY1-C’s ﬁve-stranded b-sheet overlaps with the corresponding strands of GCM with unexpected high accuracy (Figure 7A), except for the b2–b3 region including the ﬂexible loop, which will be subjected to conformational changes upon DNA binding. We thus reﬁned our DNA–AtWRKY1-C complex according to the mutagen- esis results, and the resulting model is quite similar to the GCM–DNA complex in topology. A schematic description of this reﬁned model for AtWRKY1-C and DNA interactions is described as in Figure 7B. This ﬁgure summarizes all the residues that might interact with the W-box DNA, providing structural explanations Figure 7. A model of the AtWRKY1-C and W-box interaction. (A) Superimposition of AtWRKY1-C and the overlapping structure of the Drosophila GCM domain using LSQ Fit in the program O. Macromolecular structures are shown by cartoon loops, with the GCM domain colored yellow, AtWRKY1-C blue and zinc ions as orange and magenta spheres. The DNA double-helix is shown with sticks wrapped inside surface rendering. (B) The ﬁnal schematic model of the speciﬁc interaction of AtWRKY1-C with the W-box, combining all information obtained from the structural and mutagenesis experiments presented in this work. for the function transcription factors. ACCESSION NUMBER of the WRKY superfamily of The Protein Data Bank accession number for the AtWRKY1-C crystal structure discussed in this paper is PDB ID 2AYD. ACKNOWLEDGEMENTS We thank Dr Xinnian Dong for providing NPR1-H seeds, Dr Frederick M.Ausubel for npr1-3 seeds, Dr Biao Ding for pRTL2:dGFP (dimeric GFP); Ms Huanhuan Liang is acknowledged for her help with CD spectrum measure- ment. We also thank the beamline staﬀ (Drs Yu-Hui Dong and Peng Liu) at the Beijing Synchrotron Radiation Facilities for help with data collection. This work was