1150 Nucleic Acids Research, 2007, Vol. 35, No. 4 the lowest B-factors, we thus name this constellation as D308-W312-K341 triad. In the NMR studies, the interactions around D308 could not be seen due to the undeﬁned N-terminal of that structure, only the hydrophobic interaction of AtWRKY4-C W414-K443 (AtWRKY1-C residues W312-K341), which is part of the features of the previously deﬁned triad constellation, has been mentioned (31). Figure 3D depicts another well conserved salt-bridge between Asp304 and Arg345 in the N-terminal end of the loop, and Arg345 is hydrogen bonded with the main chain of Thr301, Phe303 and Thr350. The zinc-binding site in AtWRKY1-C One of the important features resolved by the high-resolution crystal structure of AtWRKY1-C is the well-ordered zinc-binding site. As shown in the electron density map in Figure 4A, the high-resolution data of 1.6 A˚ made it possible to distinguish the zinc coordination with all the ligand atoms, and it was clearly shown that the Nd of His361 and the Ne of His363, coordinate to the zinc ion (Figure 4A and B). The distances between the ligand atoms and the zinc ion are 2.06 A˚ (Zn–Nd of His361), 2.07 A˚ (Zn–Ne of His363), 2.28 A˚ (Zn–Sg of Cys332), 2.27 A˚ (Zn–Sg of Cys337), all the distances are well within the ideal distances of a zinc-binding site in proteins (49). The whole zinc-binding site is situated in the low B-factor regions (Figure 3), and the zinc ligand residues have almost the lowest B-factors in the structure, the zinc-binding site is right next to the low B-factor D308-W312-K341 triad described in the Figure 3C. Together with the D308-W312-K341 triad, the zinc- binding site is shaping up and stabilizing the AtWRKY1-C structure, since the zinc binding has been shown to be crucial for the stability of WRKY proteins (31). The sequence signature of this zinc-binding site, C–X4–C–X23–H–X–H, somewhat resembles a classic C2H2 proﬁle (50), the feature of a zinc-ﬁnger. In the crystal structure, it has been clearly shown that the zinc ion is coordinated by two cysteines (Cys332, Cys337) and two histidines (His361, His363) (Figure 4), but structurally dissimilar to classic zinc ﬁngers, instead the structure is similar to a sequence unrelated DNA-binding protein Drosophila GCM (glia cell missing) (44) (PDB ID 1ODH). In contrast with the classic C2H2 zinc ﬁnger, AtWRKY1-C does not contain any helix, so it cannot bind DNA through the helix as most zinc-ﬁnger proteins do. The DNA-binding ability of AtWRKY1-C is mediated through the beta-hairpin regions between b2 and b3, and this mode of DNA–protein interaction is similar to the insightful prediction by Church, Sussman and Kim long time ago for non-helical DNA binding (51). Previous work on DNA binding of WRKY family has also pointed out that the conserved WRKYGQK region located on b2 strand is important for DNA binding (29). Figure 4. The zinc coordination environment. (A) 3Fo-2Fc electron- density map. The coordination of the zinc ion by Cys332, Cys337, His361 and His363 was shown. The map was prepared using CCP4 program suite and displayed with Pymol at the contour of 1.5 level. The zinc ion is shown as a purple sphere and the zinc-coordinating residues are represented by stick (yellow for C, red for O, blue for N, orange for S, respectively). (B) The distances between the zinc ion and the coordinate residues. The orientation and colors are the same as shown in Figure 4A. Conserved residues responsible for DNA binding There are more than 70 members in the Arabidopsis WRKY family grouped into three major groups (26), nine members from the group I with two WRKY domains are selected for multiple sequence alignment based on secondary structure information (Figure 2D) (sequence data were downloaded from http://rarge.gsc.riken.go.jp/ rartf/tf_info/fasta_p1/TF_9_P1.fasta). It was shown from the alignment that at least 18 residues are strictly conserved, including the four residues coordinating with the zinc ion, and three residues in the conserved D308-W312-K341 triad. As shown in Figure 2D, more than 10 well-conserved residues are positioned in the region between b2 and b3 including the WRKYGQK motif. Particularly, the ﬁve consecutive residues RKYGQ, distinct components of the WRKY family scaﬀold, belong to the b2 strand; while the other ﬁve conserved residues located on b3 strand, in the pattern of PRSYYR/K. Considering the speciﬁc DNA sequence that all WRKY members could recognize, these conserved residues are good candidates for the speciﬁc DNA-binding process. As shown in Figure 2C, the surface charge distribution of AtWRKY1-C was uneven, with a calculated PI (point of iso-electricity) of 9.5. On one side, there were consecutive regions of positively charged residues, includ- ing Arg313, Lys314, Lys318, Lys321, Arg327 and Arg331, all belonging to the strictly conserved residues and located on b2 and b3 strands. Well-conserved Lys340 located at the beginning of strand b4 also belongs to this positively charged region. Since the WRKYGQK sequence falls nicely into this positively charged region and it has been shown that the WRKY motif is responsible to bind to the W-box (29), it can thus be deduced that the b2 and b3 strands are most likely to be involved in the process of DNA binding. Identification and confirmation of the DNA-binding residues After searching for similar structures of AtWRKY1-C using the DALI server (52), a DNA–protein complex