No abstract

The WRKY proteins comprise a major family of transcription factors that are essential in pathogen and salicylic acid responses of higher plants as well as a variety of plant-specific reactions. They share a DNA binding domain, designated as the WRKY domain, which contains an invariant WRKYGQK sequence and a CX 4-5 CX 22-23 HXH zinc binding motif. Herein, we report the NMR solution structure of the C-terminal WRKY domain of the Arabidopsis thaliana WRKY4 protein. The structure consists of a four-stranded b-sheet, with a zinc binding pocket formed by the conserved Cys/His residues located at one end of the b-sheet, revealing a novel zinc and DNA binding structure. The WRKYGQK residues correspond to the most N-terminal b-strand, kinked in the middle of the sequence by the Gly residue, which enables extensive hydrophobic interactions involving the Trp residue and contributes to the structural stability of the b-sheet. Based on a profile of NMR chemical shift perturbations, we propose that the same strand enters the DNA groove and forms contacts with the DNA bases.

The B3 DNA binding domain is shared amongst various plant-specific transcription factors, including factors involved in auxin-regulated and abscisic acid-regulated transcription. Herein, we report the NMR solution structure of the B3 domain of the Arabidopsis thaliana cold-responsive transcription factor RAV1. The structure consists of a seven-stranded open b-barrel and two a-helices located at the ends of the barrel and is significantly similar to the structure of the noncatalytic DNA binding domain of the restriction enzyme EcoRII. An NMR titration experiment revealed a DNA recognition interface that enabled us to propose a structural model of the protein-DNA complex. The locations of the DNA-contacting residues are also likely to be similar to those of the EcoRII DNA binding domain.

Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). In this study, we achieved the full productivity of cell-free protein synthesis for difficult, bulky non-canonical amino acids, such as N ε-((((E)-cyclooct-2-en-1-yl)­oxy)­carbonyl)-l-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS. First, based on the crystal structure of M. alvus PylRS, the productivities for various non-canonical amino acids were greatly increased by rational engineering of the amino acid-binding pocket. The productivities were further enhanced by using a much higher concentration of PylRS over that of M. mazei PylRS, or by mutating the outer layer of the amino acid-binding pocket. Thus, we achieved full productivity even for TCO*Lys. The quantity and quality of the cell-free-produced antibody fragment containing TCO*Lys were drastically improved. These results demonstrate the importance of full productivity for the expanded genetic code.

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique ␣/␤ sandwich fold composed of two ␣ helices and four antiparallel ␤ strands and has a characteristic turn named the TY-turn between ␣1 and ␣2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the ␤ sheet surface, which may be functionally important. The putative epitope (residues 58 -77) for anti-MUC16 antibodies is located around the ␤2 and ␤3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting ␤2 and ␤3.CA125 is a serum marker that is widely used to monitor ovarian cancer because it is overexpressed in ovarian cancer cells and secreted into the blood. An elevated serum CA125 level is a useful indicator of ovarian cancer, but it is also observed in a number of benign conditions (1, 2). CA125 is a mucin-type O-linked glycoprotein (3, 4), but other details about its molecular nature remain unclear. Recently two research groups cloned CA125 (5-8), revealing that CA125 is a membrane protein with some splicing variants. The splicing variants have the same intracellular and transmembrane regions. The extracellular domain consists of the SEA 1 domains, which are repeated 7, 12, or 60 times, according to the variant. The gene was named MUC16, after the mucin-like nature of CA125. The elucidation of the amino acid sequence has made it possible to specify the approximate position of the epitope. A previous study showed that the peptide epitope position of CA125 is located between two conserved cysteines in the SEA domain (7).A cDNA of the murine MUC16 homologue, cloned in the RIKEN FANTOM project (9), has a total of 258 amino acids and a transmembrane domain. It is 66% identical to the C terminus of human MUC16 and has only one SEA domain in its extracellular region. However, our investigation of the mouse and human genomic sequences showed that they share the same characteristic repeat structure of MUC16. Thus, the murine MUC16 appears to have splicing variants, as i...