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

Abstract

Human MTH1 (hMTH1) is an enzyme that hydrolyses several oxidized purine nucleoside triphosphates to their corresponding nucleoside monophosphates. Crystallographic studies have shown that the accurate mode of interaction between 8-oxoguanine and hMTH1 cannot be understood without determining the positions of the H atoms, as can be observed in neutron and/or ultrahigh-resolution X-ray diffraction studies. The hMTH1 protein prepared in the original expression system from Escherichia coli did not appear to be suitable for obtaining high-quality crystals because the hMTH1 protein had heterogeneous N-termini of Met1 and Gly2 that resulted from N-terminal Met excision by methionine aminopeptidase from the E. coli host. To obtain homogeneous hMTH1, the Gly at the second position was replaced by Lys. As a result, mutant hMTH1 protein [hMTH1(G2K)] with a homogeneous N-terminus could be prepared and high-quality crystals which diffracted to near 1.1 Å resolution using synchrotron radiation were produced. The new crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.36, b = 47.58, c = 123.89 Å.

Structural and Kinetic Studies of the Human Nudix Hydrolase MTH1 Reveal the Mechanism for Its Broad Substrate Specificity

“…We previously reported that hMTH1(G2K) (it has a homogeneous N terminus) shows a new crystal form with high diffraction quality (1.2 Å) at neutral pH. In addition, the catalytic activity of hMTH1(G2K) toward 8-oxo-dGTP is almost identical to that of the wild type ( 32 ). Hereafter, hMTH1(G2K) is referred to as hMTH1 or the wild type for simplicity.…”

“…D120N or D120A mutations were introduced using the KOD-Plus-Mutagenesis Kit (Toyobo). The pET8c plasmid encoding hMTH1(G2K) served as a template ( 32 ). PCRs were performed with appropriate primers: 5′- AAC AGCTACTGGTTTCCACTCCTGCT-3′ for the D120N mutant and 5′- CAG CTACTGGTTTCCACTCCTGC-3′ for the D120A mutant (the mutation sites are underlined), with 5′-ATCGGGCCACATGTCCTTGAAGGG-3′ as a general reverse complementary primer for both mutants.…”

“…The hMTH1(G2K/C87A/C104S) mutant gene was synthesized by consideration of E. coli codon usage and cloned into the NdeI and XhoI sites of pET24a. The expression and purifications of hMTH1(G2K), hMTH1(G2K/D120N), hMTH1(G2K/D120A), and hMTH1(G2K/C87A/C104S) were conducted as described elsewhere ( 32 ). The purified proteins were concentrated to 10 mg/ml in a buffer consisting of 20 m m HCl, pH 7.5, 1 m m EDTA, 5% glycerol, and 1 m m β-mercaptoethanol.…”

“…Here, we report the crystal structures of hMTH1 at neutral pH in complex with the major substrates, 8-oxo-dGTP and 2-oxo-dATP, at 1.21- and 1.20-Å resolution, respectively, using our hMTH1(G2K) mutant (it contains a homogeneous N terminus), which produces a new crystal form while retaining the hydrolytic activity ( 32 ). These crystal structures showed clear electron densities of the ligands, including the triphosphate moiety that bind to the Nudix motif (hydrolase motif) and align for the hydrolysis reaction.…”

Biological characterisation and application of human MTH1 and monoclonal antibody preparation

“…It was shown that MTH1 inhibitors, such as TH287, TH588 and (S)-crizotinib, were sufficient to produce DNA breaks and induce tumour suppressor responses (13,17). Researchers have expressed recombinant human MTH1 protein in Escherichia coli for crystallisation (18)(19)(20) and inhibition experiments (13,17,21). However, there have been few reports on the optimised expression of hMTH1, as well as its purity and enzymatic activity, which are key factors for inhibitor screening and the functional analysis of hMTH1…”