High-resolution X-ray Crystallographic Study on the Enzyme Associated with Cancer — For Elucidation of the Reaction and Inhibitor Binding Mechanisms at Sub-atomic Resolution —

DNA, which carries genetic information, is damaged by ultraviolet light, chemicals and reactive oxygen species. Living organisms have mechanisms to prevent and repair the DNA damages, and maintain the genetic information. Degradation of oxidatively damaged nucleotides is one of the mechanisms of DNA damage avoidance because oxidative damage of DNA is accumulated by misincorporation of the oxidized nucleotides into DNA.

In human, MTH1, which hydrolyzes several oxidized nucleotides with its broad substrate specificity, prevents accumulation of oxidative damage in DNA and suppresses mutations. X-ray crystallography is essential for understanding the broad substrate specificity of MTH1. While MTH1 suppresses mutations, MTH1 is highly expressed in cancer cells. In 2014, research articles in Nature showed that MTH1 inhibition reduces survival of cancer cells. Now human MTH1 is highlighted as a potential anticancer target, and the efficacy of MTH1 inhibition in cancer cells is still examined by various research groups.

We participated in the High-Quality Protein Crystal Growth project operated by JAXA while examining the conditions to prepare large crystals on the ground. The project members evaluated the monodispersity, carried out further purification, and optimized the crystallization conditions using our samples. Through these experiments, the purification protocol for highly pure MTH1 was also established. Although microseeding is essential for the crystallization of MTH1, MTH1 was successfully crystallized under microgravity because a microseeding method in the space experiments has already been established.

The space experiment produced high-quality crystals that diffracted X-rays to 1.04-Å resolution (Figure 1), the best resolution at that point, and the X-ray structure was refined (Figure 2). As stated above, we have suggested that the two Asp residues in the active site vary their protonation states through binding of substrates and inhibitors. Protonation states of the carboxyl group of Asp and Glu residues can be determined by analysis of the C-O bond lengths of the carboxyl group using high-resolution structures at ~1.0-Å resolution. Using the 1.04 Å resolution data obtained in the space experiments, we analyzed the bond lengths of the Asp residues. The C-O bond lengths of Asp119 are 1.206 (15) Å and 1.311 (15) Å, respectively, and those of Asp120 are 1.236 (13) Å and 1.264 (14) Å, respectively. This result indicated that Asp119 is protonated and Asp120 is deprotonated for recognition of a substrate (8-oxo-dGTP).

This study was conducted in collaboration with Dr. Yuriko Yamagata, President of Shokei University and Shokei Junior College and Emeritus Professor of Kumamoto University.

In order to validate mechanisms of enzyme reactions, the structure determination of all atoms including hydrogens is essential. We are working on researches with the experience in this project.