Tomoko Yamasaki

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.

Structural Basis for Sequence-specific DNA Recognition by an Arabidopsis WRKY Transcription Factor

Background: The WRKY transcription factors recognize the W-box DNA element in target genes. Results: We determined the NMR solution structure of the WRKY DNA-binding domain of Arabidopsis WRKY4 in complex with W-box DNA. Conclusion: Apolar contacts by residues in the conserved WRKYGQK motif with thymine methyl groups are important in recognition of the W-box sequence. Significance: This is the first structure of the WRKY-DNA complex. The WRKY family transcription factors regulate plant-specific reactions that are mostly related to biotic and abiotic stresses. They share the WRKY domain, which recognizes a DNA element (TTGAC(C/T)) termed the W-box, in target genes. Here, we determined the solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA by NMR. A four-stranded β-sheet enters the major groove of DNA in an atypical mode termed the β-wedge, where the sheet is nearly perpendicular to the DNA helical axis. Residues in the conserved WRKYGQK motif contact DNA bases mainly through extensive apolar contacts with thymine methyl groups. The importance of these contacts was verified by substituting the relevant T bases with U and by surface plasmon resonance analyses of DNA binding.

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 N-terminal domain of the archaeal D-family DNA polymerase small subunit reveals evolutionary relationship to eukaryotic B-family polymerases

Archaea‐specific D‐family DNA polymerase forms a heterotetramer consisting of two large polymerase subunits and two small exonuclease subunits. We analyzed the structure of the N‐terminal 200 amino‐acid regulatory region of the small subunit by NMR and revealed that the N‐terminal ∼70 amino‐acid region is folded. The structure consists of a four‐α‐helix bundle including a short parallel β‐sheet, which is similar to the N‐terminal regions of the B subunits of human DNA polymerases α and ε, establishing evolutionary relationships among these archaeal and eukaryotic polymerases. We observed monomer–dimer equilibrium of this domain, which may be related to holoenzyme architecture and/or functional regulation.

Thermal denaturation of a recombinant mouse amelogenin: Circular dichroism and differential scanning calorimetric studies

Conformational analyses of a recombinant mouse tooth enamel amelogenin (rM179) were performed using circular dichroism (CD), fluorescence, differential scanning calorimetry, and sedimentation equilibrium studies. The results show that the far‐UV CD spectra of rM179 at acidic pH and 10°C are different from the spectra of random coil in 6 M GdnHCl. A near‐UV CD spectrum of rM179 at 10°C is similar to that of rM179 in 6 M GdnHCl, which indicates that aromatic residues of native structure are exposed to solvent and rotate freely. Far‐UV CD values of rM179 at 80°C are different from that of random‐coil structure in 6 M GdnHCl, which suggests that rM179 at 80°C has specific secondary structures. A gradual thermal transition was observed by far‐UV CD, which is interpreted as a weak cooperative transition from specific secondary structures to other specific secondary structures. The fluorescence emission maximum for the spectrum due to Trp residues in rM179 at 10°C shows the same fluorescence emission maximum as rM179 in 6 M GdnHCl and amino acid Trp, which indicates that the three Trp in rM179 are exposed to solvent. Deconvolution of differential scanning calorimetry curve gives the population of three states (A, I, and C states). These results indicate that three states (A, I, and C) have specific secondary structures, in which hydrophobic and Trp residues are exposed to the solvent. The thermodynamic characteristics of rM179 are unique and different from a typical globular protein, proline‐rich peptides, and a molten globule state. Proteins 2006.

Real-time NMR monitoring of protein-folding kinetics by a recycle flow system for temperature jump.

An NMR method was developed that allows for real-time monitoring of reactions (on the order of seconds) induced by a temperature jump. In a recycle flow system, heating and cooling baths were integrated, with the latter inside the NMR probe. A refolding reaction of ribonuclease A was triggered by rapid cooling and monitored by a series of NMR measurements over 12 s. Data were processed by principal component analysis, in which a factor related to the structural change with an exponential rate constant of 0.2-0.7 s(-1) was successfully separated from factors related to baseline instability and/or noise. Temperature dependency of the rate constant revealed the entropy-driven formation of the transition state of the refolding reaction.

Heat denaturation and cold denaturation of Escherichia coli RNase HI investigated by circular dichroism

Abstract Circular dichroism has been used to investigate the thermodynamics of the thermal unfolding of Escherichia coli ribonuclease HI as a function of pH over the pH range 0–4. This protein undergoes a reversible thermal conformational change from the native state to the denatured state with an isodichroic point. The calculated thermodynamic values at pH 3.0 are as follows: tm = 50.2°C, ΔHm (tm) = 93.8 kcal mol−1, ΔG (25°) = 6.08 kcal mol−1, and ΔG (10°C) = 8.14 kcal mol−1. At pH 4, ΔG (25°C) = 10 kcal mol−1 and ΔG (10°C) = 12 kcal mol−1. At a pH below 2, this protein denatures at 25°C with ΔG (25°C) = −1 kcal mol−1, but it is stable at 10°C with ΔG (10°C) = 2 kcal mol−1. The ΔCp value determined from the ΔHm(Tm) versus Tm plot is 1.4 kcal mol−1 K−1. The thermal unfolding curves at pH values above 2.18 showed a highly cooperative thermal transition. Cold denaturation was observed at temperatures below 10°C between pH 2.03 and 1.55. A cooperative heat denaturation was also observed over this pH range. At pH values lower than 1.45, cold denaturation was not observed. A broad thermal transition was observed between pH 0.83 and 0.53.

The trimeric solution structure and fucose-binding mechanism of the core fucosylation-specific lectin PhoSL.

The core α1–6 fucosylation-specific lectin from a mushroom Pholiota squarrosa (PhoSL) is a potential tool for precise diagnosis of cancers. This lectin consists of only 40 amino acids and can be chemically synthesized. We showed here that a synthesized PhoSL peptide formed a trimer by gel filtration and chemical cross-linking assays, and determined a structure of the PhoSL trimer by NMR. The structure possesses a β-prism motif with a three-fold rotational symmetry, where three antiparallel β-sheets are tightly connected by swapping of β-strands. A triad of Trp residues comprises the structural core, forming NH–π electrostatic interactions among the indole rings. NMR analysis with an excess amount of fucose revealed the structural basis for the molecular recognition. Namely, fucose deeply enters a pocket formed at a junction of β-sheet edges, with the methyl group placed at the bottom. It forms a number of hydrophobic and hydrogen-bonding interactions with PhoSL residues. In spite of partial similarities to the structures of other functionally related lectins, the arrangement of the antiparallel β-sheets in the PhoSL trimer is novel as a structural scaffold, and thus defines a novel type of lectin structure.