WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental 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 Å resolution has revealed that this domain is composed of a globular structure with five β strands, forming an antiparallel β-sheet. A novel zinc-binding site is situated at one end of the β-sheet, between strands β4 and β5. 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 β2 and β3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain and muscle ARNT-like 1) are both transcription factors of the circadian core loop in mammals. Recently published mouse CLOCK-BMAL1 bHLH (basic helix-loop-helix)-PAS (period-ARNT-single-minded) complex structure sheds light on the mechanism for heterodimer formation, but the structural details of the protein-DNA recognition mechanisms remain elusive. Here we have elucidated the crystal structure of human CLOCK-BMAL1 bHLH domains bound to a canonical E-box DNA. We demonstrate that CLOCK and BMAL1 bHLH domains can be mutually selected, and that hydrogen-bonding networks mediate their E-box recognition. We identified a hydrophobic contact between BMAL1 Ile80 and a flanking thymine nucleotide, suggesting that CLOCK-BMAL1 actually reads 7-bp DNA and not the previously believed 6-bp DNA. To find potential non-canonical E-boxes that could be recognized by CLOCK-BMAL1, we constructed systematic single-nucleotide mutations on the E-box and measured their relevant affinities. We defined two non-canonical E-box patterns with high affinities, AACGTGA and CATGTGA, in which the flanking A7-T7′ base pair is indispensable for recognition. These results will help us to identify functional CLOCK-BMAL1-binding sites in vivo and to search for clock-controlled genes. Furthermore, we assessed the inhibitory role of potential phosphorylation sites in bHLH regions. We found that the phospho-mimicking mutation on BMAL1 Ser78 could efficiently block DNA binding as well as abolish normal circadian oscillation in cells. We propose that BMAL1 Ser78 should be a key residue mediating input signal-regulated transcriptional inhibition for external cues to entrain the circadian clock by kinase cascade.

Adenylate kinases (AKs) play important roles in nucleotide metabolism in all organisms and in cellular energetics by means of phosphotransfer networks in eukaryotes. The crystal structure of a human AK named AK6 was determined by in-house sulfur single-wavelength anomalous dispersion phasing methods and refined to 2.0-Å resolution with a free R factor of 21.8%. Sequence analyses revealed that human AK6 belongs to a distinct subfamily of AKs present in all eukaryotic organisms sequenced so far. Enzymatic assays show that human AK6 has properties similar with other AKs, particularly with AK5. Fluorescence microscopy showed that human AK6 is localized predominantly to the nucleus of HeLa cells. The identification of a nuclear-localized AK sheds light on nucleotide metabolism in the nucleus and the energetic communication between mitochondria and nucleus by means of phosphotransfer networks.x-ray crystallography ͉ nuclear localization ͉ nucleotide metabolism ͉ phosphotransfer networks N ucleoside monophosphate kinases (NMPKs), which phosphorylate nucleoside monophosphates or deoxynucleoside monophosphates, which produce nucleoside diphosphates or deoxynucleoside diphosphates, play important roles in the maintenance of intracellular nucleotide pools in all organisms. Adenylate kinases (AKs) (ATP:AMP phophotransferases, EC 2.7.4.3) catalyze the reversible transfer of the ␥-phosphate group from a phosphate donor (normally ATP) to AMP, releasing two molecules of ADP (1). Besides crucial roles in homeostasis of adenine nucleotide metabolism, AKs are involved in cellular energetics through complex phosphotransfer networks regulating intracellular ATP-producing processes (2, 3).At present, five AK isoforms with different subcellular localization and substrate specificity have been characterized in mammalian tissues (4). In this work, we have identified a sixth AK isoform through a characterization of the adrenal gland protein AD-004 gene as part of a structural genomics project involving human genes (5).The AD-004 gene was first identified in a gene expression profiling study of the human hypothalamus-pituitary-adrenal axis (6) and has also appeared in analyses of genomic and cDNA sequences (7,8). The putative protein sequence for AD-004 contains 172 residues with an expected molecular mass of 20 kDa. AD-004 shows low overall sequence identity with proteins of known structures; however, in its N-terminal regions are sequence motifs [most notably a Walker motif (9)] that are characteristic of NMPKs. Through the work presented here, including crystal structure determination, enzymatic assays, and subcellular localization experiments, we show that AD-004 constitutes a AKs isoform that has been named human AK6, with many characteristics that distinguish it from the other eukaryotic AKs. Materials and MethodsProtein Purification and Crystallization. The preparation of protein used for both enzymatic assays and crystallization trials of human AD-004 (also referred to as AK6) has been described in ref. 10. Crystals of AK6 were ob...