Crystallization and preliminary X-ray analysis of human MTH1 complexed with two oxidized nucleotides, 8-oxo-dGMP and 2-oxo-dATP

Abstract

Human MutT homologue 1 (hMTH1) hydrolyzes a variety of oxidized purine nucleoside triphosphates, including 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP and 8-oxo-dATP, to their corresponding nucleoside monophosphates, while Esherichia coli MutT possesses prominent substrate specificity for 8-oxoguanine nucleotides. Three types of crystals were obtained corresponding to the following complexes: selenomethionine-labelled hMTH1 with 8-oxo-dGMP (SeMet hMTH1-8-oxo-dGMP), hMTH1 with 8-oxo-dGMP (hMTH1-8-oxodGMP) and hMTH1 with 2-oxo-dATP (hMTH1-2-oxo-dATP). Crystals of the SeMet hMTH1-8-oxo-dGMP complex belong to space group P4 1 2 1 2, with unitcell parameters a = b = 45.8, c = 153.6 Å , and diffracted to 2.90 Å . Crystals of hMTH1-8-oxo-dGMP and hMTH1-2-oxo-dATP belong to space groups P2 1 and P2 1 2 1 2 1 , with unit-cell parameters a = 34.0, b = 59.0, c = 65.9 Å , = 90.7 and a = 59.2, b = 67.3, c = 80.0 Å , respectively. Their diffraction data were collected at resolutions of 1.95 and 2.22 Å , respectively.

Human MTH3 (NUDT18) Protein Hydrolyzes Oxidized Forms of Guanosine and Deoxyguanosine Diphosphates

“…Furthermore, the 8-oxoGua base-binding mode and the glycosidic conformation are quite different in MutT and MTH1, although the sugar and phosphate positions of the two 8-oxo-dGMPs are similar. In contrast to the finding that MutT and MTH1 exist as monomers (32,34,36), NUDT5 forms a homodimer with substantial domain swapping (33,35). In NUDT5, the nucleoside moiety of 8-oxo-dGDP binds to a completely different region near the dimer interface without ligand-induced conformational changes.…”

“…The substrate recognition and binding may be achieved by a few amino acid residues located near or within the active center, and thus, elucidation of the three-dimensional structures is essential for solving this problem. Analyses of the crystal structures of MutT, MTH1, and NUDT5, all of which are complexed with 8-oxo-Gua-containing nucleotides, have been performed (32)(33)(34)(35)(36)(37). In the cases of MutT and MTH1, the reaction product 8-oxo-dGMP binds to the same substrate- binding pocket composed of two ␤-sheets and one ␣-helix.…”

Structural and Dynamic Features of the MutT Protein in the Recognition of Nucleotides with the Mutagenic 8-Oxoguanine Base

“…Native MutT was overexpressed in Luria-Bertani (LB) broth, and SeMet MutT was overexpressed in LeMaster broth containing seleno-DL-methionine instead of methionine with sufficient amounts of isoleucine, lysine, and threonine to inhibit the methionine pathway (20,21). This condition was also present in the overexpression of SeMet hMTH1 (22). Purification of MutT was carried out by almost the same procedure (except that the hydroxyapatite column chromatography step was skipped), as described previously (23).…”

Crystallization and preliminary X-ray analysis of human MTH1 with a homogeneous N-terminus

“…Previously, we reported the solution structure of hMTH1 and the substrate-binding region using the NMR method and the preliminary X-ray analyses of hMTH1 in complexes with 8-oxo-dGMP and 2-oxo-dATP (Mishima et al, 2004;Nakamura et al, 2006). Recently, crystal structures of hMTH1 with and without 8-oxo-dGMP have been reported (Svensson et al, 2011).…”

“…These results revealed that hMTH1 binds 8-oxo-dGMP without any conformational change and that Asp119 or Asp120 needs to be protonated for recognition of the 8-oxoguanine base in the complex of hMTH1 with 8-oxo-dGMP. These crystals were grown at pH 4.0 (Nakamura et al, 2006;Svensson et al, 2011). Therefore, the crystallization of hMTH1 complexed with substrate at a neutral pH is required.…”