structure of AtWRKY1-C looks rather globular and stable (Figure 2A). The crystal structure of AtWRKY1-C diﬀers from the NMR structure of a similar protein AtWRKY4 C- terminal domain (AtWRKY4-C, which shares about 53% sequence identity with AtWRKY1-C) determined recently (31). As shown in Figure 2B and D, the b1 strand of AtWRKY1-C was missing in the NMR AtWRKY4-C model, thus the strands b2 to b5 in the crystal structure of AtWRKY1-C corresponding to the strands b1–b4 of the NMR structure, and the RMSD corresponding to the similar regions of the two structures (AtWRKY1-C residues 309–367, AtWRKY4-C residues 411–469) is 1.5 A˚, indicating a very similar structure for a common DNA-binding mechanism. Since the AtWRKY4-C domain was constructed to start from VQTTS which is located in the middle of the b1 strand (Figure 2B and D with the alignment), the discrepancy in the organization of the b-sheet is most likely caused by the diﬀerent ways of preparing the NMR and crystallization samples. In order to have a fair comparison, we have repeated and constructed the NMR samples in this work and compared the CD spectra for the two proteins, AtWRKY4-C (Asp365–Ala469) similarly constructed as AtWRKY1-C, and AtWRKY4-C 1b (Val399–Ala469) similar to the NMR sample without the N-terminal b1 strand. The CD spectra showed similar pattern for zinc binding for both AtWRKY4-C and AtWRKY4-C 1b, and characteristics in both cases of a folded b-sheet structure. However, there was a reduction in the CD signal of AtWRKY4- C 1b near the negative peak at 210–220 nm, indicating a reduction of b-sheet signal of the truncated protein (data not shown). We thus predict that the AtWRKY4-C would have a similar globular structure like AtWRKY1-C, had the AtWRKY4-C sample prepared several residues longer at the N-terminus. The conserved b1 and B-factor distribution of AtWRKY1-C As shown in Figure 2D, in addition to the conserved WRKY deﬁning residues (in red, yellow and blue), the sequences in the b1 region (colored green) are well conserved in the C-terminal domains of the group I WRKY family members. We can thus conclude that all the C-terminal domains of the group I WRKY protein possess a very similar ﬁve-stranded b-sheet architecture as in AtWRKY1-C. Figure 3 describes the B-factor distribution over the AtWRKY1-C structure. Figure 3A shows the B-factor plot against the residue numbers, and Figure 3B is the overall structure overlaid with the B-factors color coded as deﬁned in Figure 3A. The regions with high B-factors (higher than 25) are in both the N- and C-terminal ends and on the loop between b2 and b3, this loop may be involved in conformational changes upon DNA binding. The middle part of the long loop connecting b1 and b2, and a few residues after b5 also showed slightly high B-factor between 20 and 25, whereas most of the structure are quite stable and ordered with an average B-factor of 21, particularly the zinc-binding site and the central b sheet are all with B-factors below 20. Nucleic Acids Research, 2007, Vol. 35, No. 4 1149 Figure 3. B-factor representation of AtWRKY1-C crystal structure. All the objects are colored according to the B-factor values, from blue to red in the order of increasing B-factor. (A) Plot of B-factor against residues, with B-factor averaged over each residue. (B) The overall structure of AtWRKY1-C in ribbon colored according to the B-factor values. The ends of the loop between strands b1 and b2 with low B-factor values are circled out by red rings for further ampliﬁcation in 3C and 3D. Details of the interaction around these two terminals are described in 3C and 3D. (C) A D308-W312-K341 triad in the C-terminal of the loop between b1 and b2. Asp308 forms a well-deﬁned salt-bridge with Lys341, extensively H-bonding with side chains of Trp312, Tyr357 and backbone of Tyr310. The three key residues Asp308, Trp312 and Lys341, components of D308-W312-K341 triad, and their hydrogen bonded residues Gly309, Tyr357 are marked by stars in Figure 2D. (D) The stable N-terminal end of the loop between b1 and b2. Arg345 is hydrogen bonded with the backbones of Thr301, Phe303 and Thr350. A potential salt-bridge between Arg345 and Asp304 was also observed as shown, and these two conserved residues are marked by triangles in Figure 2D. The conserved salt-bridges and the D308-W312-K341 triad Interestingly, both ends of the connecting loop between b1 and b2 show conserved pattern with ordered salt-bridge interactions and extensive hydrogen-bonding networks (Figure 3C and D). Figure 3C shows that in the C-terminal end of the loop, Asp308 forms a well-deﬁned salt-bridge with Lys341, and Asp308 also forms extensive H-bonds with Tyr310, Trp312, Tyr357. All these residues are very well conserved among the WRKY family proteins, indicating a common function of stabilizing the domain structure. The pivotal residue at this constellation of residues is Asp308. The distance between Asp308 OD1 and Trp312 NE1 is 2.94 A˚ , and between Asp308 OD2 and Lys341 NZ is 2.85 A˚ . These three residues are with