Purification, Crystallization, and Preliminary Crystallographic Studies of Human As(III) S-Adenosylmethionine Methyltransferase (hAS3MT)

Abstract

Exposure to environmental arsenic is associated with serious of health issues such as cancer, diabetes and developmental delays in infants and children. In human liver, As(III) S-adenosylmethionine methyl transferase (hAS3MT) (EC 2.1.1.137) was proposed to be an detoxification process by methylation of inorganic arsenite into pentavalent methyl MAs(V) and dimethyl arsenite DMAs(V). More recently the first product was shown to be highly toxic and potentially carcinogenic trivalent methylarsenite (MAs(III)). Our studies are designed to elucidate the mechanism of AS3MT and its contribution to arsenic-related diseases. Here, we report the first crystallization and preliminary X-ray diffraction analysis of the human AS3MT enzyme. The crystals belong to the monoclinic P12₁1 space group with unit cell parameters of a = 135.03 Å, b = 260.44 Å, c = 279.03 Å, α = 90.00°, β = 93.36°, γ = 90.00°.

INTRODUCTION

Arsenic is a toxic metalloid found ubiquitously in the environment. On the 2019 list of the most hazardous substances provided by the U.S Environmental Protection Agency (EPA) and Agency for Toxic Substances and Disease Registry (ATSD), arsenic is at the top (https://www.atsdr.cdc.gov/spl/index.html#2019spl). The International Agency on Research Cancer also lists both inorganic and organic forms of arsenic as human carcinogens (https://monographs.iarc.fr/agents-classified-by-the-iarc/). From bacteria to humans, every organism is exposed to arsenic from natural environmental sources, including soil, water, food and anthropogenic sources such as herbicides and antimicrobial growth promoters [1]. Arsenicals leach out of bedrock to water sources and reach agricultural land. The World Health Organization (WHO) has warned that millions of people worldwide are exposed to arsenic from food and water supplies [2].

In members of every kingdom the enzyme S-adenosylmethionine (SAM) methyl transferase (EC 2.1.1.137) transfers the methyl group of SAM to inorganic As(III), producing methylated species. The enzyme has been termed ArsM in microbes [3] and AS3MT in animals [4]. In microorganisms methylation transforms inorganic arsenic into the highly toxic trivalent species methylarsenite (MAs(III) and dimethylarsenite (DMAs(III)), which have been proposed to be primordial antibiotics [1]. In humans, the liver enzyme, hAS3MT, activation of arsenic into toxic and potentially carcinogenic MAs(III) and DMAs(III) is proposed to be a major step in progression to arsenic-related diseases [5].

Structural information is necessary to understand the mechanism of catalysis on the one hand and to design inhibitors and modulators on the other hand that might prevent or ameliorate arsenic toxicity/carcinogenicity. The crystal structure of the heat-stable ortholog CmArsM from the thermophilic alga Cyanidioschyzon merolae has been useful for modeling the structure of hAS3MT [6], but no structural information is available for any mammalian As(III) SAM methyltransferase. The objective of the study is to elucidate structure-function relationships of human AS3MT. Here we report crystallization and preliminary X-ray diffraction data of hAS3MT, the first report of crystallization of any animal AS3MT. This information can form the basis for the discovery of small molecule modulators and subsequent structure-based drug design.

MATERIALS AND METHODS

Reagents

All reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA) unless otherwise noted. Crystallization screens were purchased from Hampton Research (Aliso Voiejo, California, USA) and Molecular Dimensions (Maumee, OH, USA).

Expression and Purification of hAS3MT

The synthetic full-length hAS3MT gene [7] was cloned as an EcoRI/SalI digest from pUC57-Kan-hAS3MT into expression vector pMAL-c2x to produce gene encoding the maltose binding protein (MBP) at the 5′ end and eight histidine residues at the 3’ end. For purification, the MBP-fused full length synthetic hAS3MT expressed in BL21(DE3) pMAL-c2x were grown at 37°C in lysogeny broth (LB) medium [8], 100 μg/mL of ampicillin, with shaking at 220 rpm. At A_600nm = 0.6, isopropyl ß-D-1-thiogalactopyranoside (IPTG) (Research Products International, Mount Prospect, Illinois, USA) was added at 0.3 mM, final concentration, to induce expression of hAS3MT. The cells were grown for another 4.5 h, harvested by centrifugation (3500g) for 20 min, washed once with buffer A (50 mM MOPS, pH 7.5, 0.3 M NaCl, containing 10 wt % glycerol and 1 mM tris(2-carboxyethyl phosphine (TCEP)) and suspended in 4 mL of buffer A per g of wet cells. The cells were lysed by a single passage through a French pressure cell at 20000 psi and immediately treated with 2.5 μL/g wet cells of the protease inhibitor diisopropylfluorophosphate (Thermofisher scientific). Membranes and unbroken cells were removed by centrifugation at 150000g for 1 h. The supernatant solution containing the MBP fusion protein was loaded onto a 5 mL amylose column at a flow rate of 1 mL/min (GE Healthcare) pre-equilibrated with buffer A using an AKTA-FPLC purification system (GE Healthcare). The column was washed with 8 column volumes of buffer A to remove unbound protein. The 85 kDa hA3MT-MBP fusion protein was eluted with buffer B (50 mM MOPS pH 7.5, 0.3 M NaCl, 10% glycerol, and 2 mM TCEP with addition of 10 mM amylose). Purity and homogeneity were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [9]. Enzyme-containing fractions were concentrated by centrifugation using a 30 kDa cutoff Amicon Ultrafilter (EMD Millipore Corporation, Billerica, Massachusetts, USA). The buffers were exchanged with buffer C (50 mM MOPS, pH 7.5, containing 0.5 M NaCl, and 2 mM TCEP) by repeated dilution with buffer C for concentration of the enzyme. The proteins were quick frozen and stored at −80°C until further use. Protein concentrations were calculated from the amino acid sequence using extinction coefficients at 280 nm of 10.1390 M⁻¹ cm⁻¹ for the full-length AS3MT-MBP chimera. To measure enzymatic activity, the assay mixture contained 1 μM purified chimeric protein, 2.5mM reduced glutathione, 10 μM thioredoxin, 1.5 μM thioredoxin reductase, 0.3 mM NADPH, and 10 μM As(GS)₃ in a buffer consisting of 50 mM NaH₂PO₄ and 0.3 M NaCl, pH 8. S-adenosylmethionine was added at a final concentration 0.5 mM to initiate the reaction at 37°C. Arsenical compounds were separated by high pressure liquid chromatography (HPLC), with arsenic concentrations determined by inductively coupled plasma mass spectrometry (ICP-MS). The activity of the purified enzyme was similar to that reported earlier [6, 7].

Crystallization

Crystallization trials were performed with protein solutions at concentrations from 9 to 29 mg/mL using hanging drop or sitting drop vapor diffusion methods at a 1 : 1 ratio of protein to reservoir solution in Limbro boxes, in buffer C (50 mM MOPS, pH 7.5, containing 0.5 M NaCl and 2 mM TCEP). Prior to each crystallization trial, the protein solutions were centrifuged for 10 min at 12000 rpm at 4°C. Experiments were performed at 18 and 20°C simultaneously with various crystal screens (Crystal Screen 1, Crystal Screen 2, PEG Ion Screen 1, PEG Ion Screen 2) from Hampton Research (Aliso Viejo, CA) and JCSG+1, JCSG+2, pH Clear Suite Screens from Molecular Dimensions (Maumee, OH). The full length MBP hAS3MT chimera at 11 mg/mL produced needle-shaped crystals in 45% MPD (2,4-methyl pentanediol), 0.2 M ammonium phosphate, 0.1 M HEPES, pH 7.5.

Data Collection and Processing

Cryoprotectant (30% ethylene glycol) was added to the crystals, which were transferred to loops and flash–frozen in liquid nitrogen for data collection. Initial screening was performed at the Southeast Regional Collaborative Access Team (SER-CAT) facility at the Advanced Photon Source (APS), Argonne, Illinois, USA. Additional data sets are collected at the Advanced Light Source, Berkeley Center for Structural Biology, CA, USA. Data were obtained from 360 image frames with 0.5° rotation angle about φ using ADSC Quantum 315r (3_3 CCD array) detector at 100 K under a liquid-nitrogen stream at ALS synchrotron beam line 8.2.1/8.2.2 with a crystal to detector distance of 300.933 mm. The data sets were indexed, integrated and scaled with the XDS suite [10], The data processing statistics are shown in Table 1.

Table 1.

X-ray data collection statistics and processing of hAS3MT

X-ray source	Synchrotron (ALS)
Wavelength, Å	1.00
Resolution range, Å	48.59–6.53
Oscillation angle, deg	0.5
Space group	P1211
a, b, c, Å	135.03, 260.44, 279.03
α, β, γ, deg	90.00, 93.36, 90.00
Crystal to detector distance, mm	300
Number of observed reflections	83376 (13371)
Number of unique reflections	27807 (4491)
Matthews coefficient, Å3 Da−1	2.38
Completeness, %	74.4 (74.4)
Multiplicity	3.0 (3.0)
〈I〉/〈σ(I)〉	5.6 (2.1)
R merge	0.231(0.887)
R pim	0.162(0.580)
Mosaicity, deg	0.38

Values in parenthesis are for highest resolution shell. The data sets were indexed, integrated and scaled with the XDS suite. R_merge = hkli Ii(hkl) – (I(hkl)) hkli Ii(hkl), where Ii(hkl) is the observed intensity and (I(hkl) is the average intensity over symmetry equivalent measurements.

RESULTS AND DISCUSSION

Synthetic human AS3MT with only a histidine tag did not produce soluble protein when expressed in E. coli. For that reason, we introduced an MBP fusion at the N-terminus and eight histidine residues at the C-terminus in vector plasmid pMAL-c2x. The synthetic construct expressed well in cells of E. coli and could be purified by affinity chromatography for crystallization trials (Fig. 1a) as a monomer (Fig. 1b). The hAS3MT protein sequence aligned with the sequence of the eukaryotic algal orthologue CmArsm (Fig. 2) with 42% identity and 59% overall sequence similarity. The construct is catalytically active, methylating As(III) to methyl and dimethyl species as described by [6].

Fig. 1.

Purification of hAS3MT. Full length hAS3MT with MBP tag purified by amylose affinity chromatography. The purity of the protein was analyzed by 10% SDS PAGE stained with Coomassie Blue (a). Size exclusion chromatography of hAS3MT shows that the protein eluted as a monomer (b).

Fig. 2.

Sequence alignment of hAS3MT (375 residues, accession number Q9HBK9) and Cyanidioschyzon sp. 5508 CmArsM (400 residues, accession number C0JV69_9RHOD). The proteins are 42% identical and 59% similar.

The hAS3MT protein initially produced needle-shaped crystals. We optimized the conditions by slight modifications of the salt concentration, resulting in three dimensional crystals of diffraction quality using the hanging drop vapor diffusion method at 18°C (Fig. 3a). The crystals diffracted up to 3.7 Å in the monoclinic P12₁1 space group (Fig. 3b) with unit cell parameters of a = 135.03 Å, b = 260.44 Å, c = 279.03 Å, α = 90.00°, β = 93.36°, γ = 90.00°. The diffraction data reduced to 6.5 Å with completeness observed to be 74.4%. The Matthews coefficient [11] suggesting that there might be 24 molecules in the crystal subunit in which each molecule is expected to be the 85 kDa MBP-AS3MT protein with Vm = 2.38 ų Da⁻¹ with 47.2% solvent content. The self-rotation function calculated using MOLREP [12], at Chi 180° indicates that there is no noncrystallographic symmetry, and Chi 90° (Fig. 3c) locate the 2-fold axis with no indication of twinning [13]. Six peaks on 30° each other were consistent with 2-fold symmetry. Further trials to obtain crystals with higher resolution are in progress, and phase information will be helpful to determine the AS3MT structure [14, 15].

Fig. 3.

Crystallization of hAS3T. Crystallization conditions: 30% MPD, 0.5 M ammonium sulfate, 0.1 M HEPES, pH 7.5. The rod-shaped crystals (approximately 0.3 × 0.5 × 0.3 mm in size) were grown at 18°C and belong to monoclinic space group P12₁1 (a). The diffraction images (0.5° oscillation) were collected from the hAS3MT crystal. The edge of the image corresponds to 3.68 Å resolution (b). Self-rotation function calculated for hAS3MT crystal data, showing the Chi at 180° and Chi at 90° sections. Peaks are represented as dense counter lines (c).