Published online 30 January 2007 Nucleic Acids Research, 2007, Vol. 35, No. 4 1145–1154 doi:10.1093/nar/gkm001 DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein Ming-Rui Duan1, 2, Jie Nan 1 , Yu-He Liang 1 , Peng Mao1, 2, Lu Lu1, 2, Lanfen Li1, Chunhong Wei1, 2, Luhua Lai3, Yi Li1, 2* and Xiao-Dong Su1, * 1The National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, P.R. China, 2Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, Beijing 100871, P.R. China and 3College of Chemistry and Chemical Engineering, Peking University, Beijing 100871, P.R. China Received August 11, 2006; Revised December 20, 2006; Accepted December 22, 2006 ABSTRACT WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified speci- fically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmen- tal 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 b strands, forming an antiparallel b-sheet. A novel zinc-binding site is situated at one end of the b- sheet, between strands b4 and b5. 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 b2 and b3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins. INTRODUCTION WRKY proteins comprise a large group of transcription factors identiﬁed speciﬁcally from the plant kingdom, ranging from lower plants such as mosses to higher plants (1,2). Members of the WRKY superfamily play important roles in a variety of developmental and physiological processes in plants. The most documented function for this superfamily of genes is their involvement in salicylic acid (SA) signaling and disease responses (3–8). SA is an important signal molecule in both local defenses and systemic acquired resistance (SAR). Endogenous accu- mulation or exogenous application of SA activates SAR and pathogenesis-related (PR) gene expression. In Arabidopsis, 49 out of 72 tested WRKY genes can be induced by pathogen infection or SA treatment (7), suggesting that a broad range of WRKY members could be involved in playing regulatory roles in disease resistance. This role of disease resistance was further conﬁrmed by the ﬁnding of an over-presentation of the cis-element activated by the WRKY proteins, the W-box, within the promoters of a number of genes that are co-expressed in SAR (9,10). Arabidopsis NON-EXPRESSOR OF PR1 (NPR1) has been demon- strated as a key protein in SAR signaling pathway (11). SAR and expression of PR1 are prevented in Arabidopsis npr1 mutants even after SA treatment (12). Previous reports also showed that over-expression of WRKY18 and WRKY70 in Arabidopsis resulted in increased resistance to pathogens and enhanced expression of PR genes (3,13). *To whom correspondence should be addressed: Tel: þ86 10 6275 9743; Fax: þ86 10 6276 5669; Email: su-xd@pku.edu.cn; Yi Li. Tel: þ86 10 6275 9690; Fax: þ86 10 6275 4427; E-mail: liyi@pku.edu.cn The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 2007 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.