Masaaki Aoki

Solution Structure of an Arabidopsis WRKY DNA Binding Domain

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 CX4–5CX22–23HXH 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 β-sheet, with a zinc binding pocket formed by the conserved Cys/His residues located at one end of the β-sheet, revealing a novel zinc and DNA binding structure. The WRKYGQK residues correspond to the most N-terminal β-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 β-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.

Solution Structure of the B3 DNA Binding Domain of the Arabidopsis Cold-Responsive Transcription Factor RAV1

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 β-barrel and two α-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.

Solution Structure of the SEA Domain from the Murine Homologue of Ovarian Cancer Antigen CA125 (MUC16)

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 α/β sandwich fold composed of two α helices and four antiparallel β strands and has a characteristic turn named the TY-turn between α1 and α2. 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 β sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the β2 and β3 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 β2 and β3.

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the α‐subunit of the only translation initiation factor (eIF2α) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N‐terminus, which is termed the RWD domain after three major RWD‐containing proteins: RING finger‐containing proteins, WD‐repeat‐containing proteins, and yeast DEAD (DEXD)‐like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an α + β sandwich fold consisting of two layers: a four‐stranded antiparallel β‐sheet, and three side‐by‐side α‐helices, with an αββββαα topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive β‐turns that overlap with each other by two residues (triple β‐turn). As putative binding sites with GCN1, a structure‐based alignment allowed the identification of several surface residues in α‐helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin‐conjugating enzymes (E2s), with most of the structural differences in the region connecting β‐strand 4 and α‐helix 3. The structural architecture, including the triple β‐turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein‐binding that plays specific roles in individual RWD‐containing proteins.

Solution structure of a BolA‐like protein from Mus musculus

The BolA‐like proteins are widely conserved from prokaryotes to eukaryotes. The BolA‐like proteins seem to be involved in cell proliferation or cell‐cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA‐like protein. The overall topology is αββααβα, in which β1 and β2 are antiparallel, and β3 is parallel to β2. This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA‐like proteins are assembled on the one side of the protein.

Solution structure of the PWWP domain of the hepatoma-derived growth factor family

Among the many PWWP‐containing proteins, the largest group of homologous proteins is related to hepatoma‐derived growth factor (HDGF). Within a well‐conserved region at the extreme N‐terminus, HDGF and five HDGF‐related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin‐associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF‐related protein‐3 (HRP‐3) by NMR spectroscopy. The structure consists of a five‐stranded β‐barrel with a PWWP‐specific long loop connecting β2 and β3 (PR‐loop), followed by a helical region including two α‐helices. Its structure was found to have a characteristic solvent‐exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the β1/β2 loop (β‐β arch) and the β3/β4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

An Arabidopsis SBP-domain fragment with a disrupted C-terminal zinc-binding site retains its tertiary structure.

SQUAMOSA promoter‐binding proteins (SBPs) form a major family of plant‐specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA‐binding domain of ∼80 amino acids (SBP domain), which contains two non‐interleaved zinc‐binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP‐domain fragment that lacks a Cys residue involved in the C‐terminal zinc‐binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA‐binding activity, we propose that the Zn2+ ion at the N‐terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C‐terminal site is necessary for the DNA binding, mainly by guiding the basic C‐terminal loop to correctly fit into the DNA groove.

Solution structure of the RNA binding domain in the human muscleblind-like protein 2.

The muscleblind‐like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1–4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre‐mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH‐type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel β‐sheet with the N‐terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three‐dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH‐type zinc finger motifs in the MBNL protein family.

Letter to the Editor: NMR assignment of the hypothetical ENTH-VHS domain At3g16270 from Arabidopsis thaliana

Blanca Lopez-Mendeza, David Pantoja-Ucedaa, Tadashi Tomizawaa, Seizo Koshibaa, Takanori Kigawaa, Mikako Shirouzua,b, Takaho Teradaa,b, Makoto Inouea, Takashi Yabukia, Masaaki Aokia, Eiko Sekia, Takayoshi Matsudaa, Hiroshi Hirotaa, Mayumi Yoshidaa, Akiko Tanakaa, Takashi Osanaia, Motoaki Sekia, Kazuo Shinozakia, Shigeyuki Yokoyamaa,b,c & Peter Gunterta,∗ aRIKEN Genomic Sciences Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan; bRIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148, Japan; cDepartment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan