structure, the Drosophila GCM (44) (PDB ID 1ODH, Z-score46.2) 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 A˚ , 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 b2 and b3. 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. As shown in Figure 5, K314A, Y315R, G316F, R331A, R327A and R327E have completely abolished the DNA-binding abilities of those mutants, and the CD spectra in Figure 6 have shown those mutants were well-structured proteins. Thus the inabilities of DNA binding must be caused by the point mutations on the correct structural scaﬀold. The DNA-binding activity of the mutations R313E, K314R, Y315F and Q317K were reduced, whereas the mutations of Q317A, R331K, K340A and Y330F did not show any decrease of the DNA-binding ability, indicating that these mutations did not aﬀect the DNA binding of AtWRKY1-C. Nucleic Acids Research, 2007, Vol. 35, No. 4 1151 Figure 5. EMSA with the consensus W-box binding site and wild-type AtWRKY1-C as well as mutant proteins. (A) Binding activity examination with mutations on b2. The binding reaction contained 5 mg puriﬁed proteins and 3 mg DNA duplex. The gel was stained with EB after the electrophoresis. The same amount of puriﬁed proteins were run on SDS–PAGE and stained with Coomassie Brilliant Blue to show the equal loading. (B) Binding activity examination with mutations on b3 and b4. These results are in good agreement with the previous work (29), and conclusions from the NMR structure (31). In the crystal structure as well as in the NMR structure, a kink was formed in the middle of b2 around Gly316. The formation of this kink was considered helpful for the protruding of Gly316 into DNA major groove deeply and attending speciﬁc binding. The assumption was conﬁrmed by the mutation G316F, which has lost almost all the DNA-binding ability. The complete loss of DNA binding of the mutants R327E, R327A and R331A are of great interests, since these two positively charged residues localized on the b3 strand that has been shown to be crucial for the DNA binding for the ﬁrst time by site-directed mutagenesis. Furthermore, these two arginine residues, which are separated by four residues, is consistent with Tateno’s model of DNA binding at the major groove with a pair of b strands (53). As a summary, all the mutation results further conﬁrmed that strands b2 and b3 participate in DNA binding. It also suggested that residues Lys314, Gly316, Lys318 on b2, and Arg327, Arg331 on b3 are the key residues for DNA-speciﬁc recognition and substitution any one of them resulted in no speciﬁc binding. In addition, the distribution of these residues is i, i þ 2, i þ 4 on one strand and j, j þ 4 on the other strand. This distribution is in agreement with Tateno’s model (53), which proposed that the binding of DNA to proteins with two b strands should happen on the DNA major groove in a convex manner. Based on such a model, it has been found that normally six consecutive base pairs are