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The structure of the DNA-binding NAC domain of Arabidopsis ANAC (abscisic-acid-responsive NAC) has been determined by Xray crystallography to 1.9 Å resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant-specific transcriptional regulators. NAC proteins are characterized by their conserved N-terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix-turnhelix motif; instead it reveals a new transcription factor fold consisting of a twisted b-sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

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NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production. The structure of the DNA-binding NAC domain of ANAC019 has previously been determined by X-ray crystallography, revealing a dimeric and predominantly β-fold structure, but the mode of binding to cognate DNA has remained elusive. In the present study, information from low resolution X-ray structures and small angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a β-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core β-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC-DNA complex and a new crystal form of the unbound NAC also indicate limited flexibility of the NAC dimer arrangement, which could be important in recognizing suboptimal binding sites.

U-box proteins, as well as other proteins involved in regulated protein degradation, are apparently over-represented in Arabidopsis compared with other model eukaryotes. The Arabidopsis protein AtPUB14 contains a typical U-box domain followed by an Armadillo repeat region, a domain organization that is frequently found in plant U-box proteins. In vitro ubiquitination assays demonstrated that AtPUB14 functions as an E3 ubiquitin ligase with specific E2 ubiquitin-conjugating enzymes. The structure of the AtPUB14 U-box domain was determined by NMR spectroscopy. It adopts the ␤␤␣␤ fold of the Prp19p U-box and RING finger domains. In these proteins, conserved hydrophobic residues form a putative E2-binding cleft. By contrast, they contain no common polar E2 binding site motif. Two hydrophobic cores stabilize the AtPUB14 U-box fold, and hydrogen bonds and salt bridges interconnect the residues corresponding to zinc ion-coordinating residues in RING domains. Residues from a C-terminal ␣-helix interact with the core domain and contribute to stabilization. The Prp19p U-box lacks a corresponding C-terminal ␣-helix. Chemical shift analysis suggested that aromatic residues exposed at the N terminus and the C-terminal ␣-helix of the AtPUB14 U-box participate in dimerization. Thus, AtPUB14 may form a biologically relevant dimer. This is the first plant U-box structure to be determined, and it provides a model for studies of the many plant U-box proteins and their interactions. Structural insight into these interactions is important, because ubiquitindependent protein degradation is a prevalent regulatory mechanism in plants.The ubiquitin proteolytic pathway plays an important role in regulated protein degradation (1). Proteins designated for degradation are covalently modified by attachment of a ubiquitin polymer and degraded by the 26 S proteasome. A ubiquitinactivating enzyme (E1) 1 catalyzes ATP-dependent formation of a thioester bond between ubiquitin and itself and transfers the activated ubiquitin to a ubiquitin-conjugating enzyme (E2). Formation of an isopeptide bond between ubiquitin and a substrate is facilitated by a ubiquitin-protein ligase (E3) that can bind both the E2-ubiquitin complex and the substrate. Members of the HECT and RING protein families are the best characterized E3 ligases, but recently U-box proteins have also been shown to function as E3s (2, 3). This may be the general function of U-box proteins, although they were initially suggested to function as ubiquitin chain assembly factors (E4s) (4). Recently, research on U-box proteins, especially the carboxyl terminus of Hsc70-interacting proteins (CHIP), has focused on their ability to interact with molecular chaperones and selectively ubiquitinate unfolded proteins. Thus ubiquitination can also function in protein quality control (5).The U-box motif is a peptide chain that contains ϳ70 amino acid residues, with characteristics suggesting that it is a structural variant of the RING fold but lacks the signature zincbinding amino acids of the RING doma...