Masaki Mishima

Paramagnetic NMR study of Cu2+–IDA complex localization on a protein surface and its application to elucidate long distance information

The paramagnetic metal chelate complex Cu2+‐iminodiacetic acid (Cu2+–IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of 1H and 15N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu2+–IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu2+ relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 Å. Here, we show that a Cu2+–IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long‐range distance information.

Identification of the core domain and the secondary structure of the transcriptional coactivator MBF1

Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ‐F1 and GCN4. MBF1 bridges the DNA‐binding regions of these activators and the TATA‐box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP.

Resonance assignments, secondary structure and 15N relaxation data of the human transcriptional coactivator hMBF1 (57-148).

Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator that is thought to bridge between the TATA box-binding protein (TBP) and DNA binding regulatory factors, and is conserved from yeast to human. Human MBF1 (hMBF1) can bind to TBP and to the nuclear receptor Ad4BP, and is suggested to mediate Ad4BP-dependent transcriptional activation. Here we report the resonance assignments and secondary structure of hMBF1 (57–148) that contains both TBP binding and activator binding residues. 15N relaxation data were also obtained. As a result, hMBF1 (57–148) was shown to consist of flexible N-terminal residues and a C-terminal domain. The C-terminal domain contains four helices and a conserved C-terminal region.

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd.