Crystallization and preliminary X-ray crystallographic analysis of human myotubularin-related protein 1

Human myotubularin-related protein 1 (MTMR1) was crystallized using polyethylene glycol 20 000 as a precipitant. Diffraction data have been collected to 2.0 Å resolution using synchrotron X-rays.

Abstract

Myotubularin-related protein 1 is a phosphatase that dephosphorylates phospholipids such as phosphatidylinositol 3-phosphate or phosphatidylinositol 3,5-bisphosphate. In this study, human MTMR1 was overexpressed in Escherichia coli, purified and crystallized at 277 K using polyethylene glycol 20 000 as a precipitant. Diffraction data were collected to 2.0 Å resolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a = 67.219, b = 96.587, c = 97.581 Å, α = 87.597, β = 86.072, γ = 77.327°. Assuming the presence of four molecules in the asymmetric unit, the calculated Matthews coefficient value was 2.61 ų Da⁻¹ and the corresponding solvent content was 52.9%.

1. Introduction  

Phosphatidylinositol (PtdIns) in eukaryotic membranes can be phosphorylated at the D3, D4 and D5 positions of the inositol ring, resulting in the formation of seven different phospho­inositides (PIs; Robinson & Dixon, 2006 ▶). PIs play important roles in signal transduction, membrane trafficking, cell growth and survival, cell division and cellular motility (Robinson & Dixon, 2006 ▶). The phosphate at the D3 position of phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] can be dephosphorylated by myotubularin and myotubularin-related proteins (MTM1 and MTMRs; Clague & Lorenzo, 2005 ▶; Robinson & Dixon, 2006 ▶).

The myotubularin family is a subgroup of the tyrosine/dual-specificity phosphatase (PTP/DSP) superfamily (Laporte et al., 2003 ▶). While the PTP domains in MTM1, MTMR1, MTMR2, MTMR3, MTMR4, MTMR6, MTMR7 and MTMR8 possess catalytically active PTP with a CX ₅R consensus motif, the other six members of the MTMRs (MTMR5 and MTMR9–MTMR13) have inactive PTP domains (Robinson & Dixon, 2006 ▶; Nandurkar & Huysmans, 2002 ▶; Laporte et al., 2003 ▶). These inactive MTMRs interact with the catalytically active MTMRs via their coiled-coil domain, regulating the function of the MTMRs (Kim et al., 2003 ▶; Hnia et al., 2012 ▶).

Human MTMR1 contains 665 amino acids, a PH-GRAM domain (Doerks et al., 2000 ▶), an active PTP domain, a coiled-coil domain and a PDZ-binding motif. Although MTMR1 was one of the first myotubularin-related proteins to be identified, it remains poorly studied. It displays high amino-acid sequence similarity to MTM1 and MTMR2. It has been reported that aberrant expression of MTMR1 leads to myotonic dystrophy (DM; Buj-Bello et al., 2002 ▶; Hnia et al., 2012 ▶).

Some structural studies on MTMRs have been performed. Crystal structures of MTMR2 have been elucidated, revealing the substrate specificity and the catalytic mechanism (Begley et al., 2003 ▶, 2006 ▶). The crystal structure of MTMR6 has also been determined and deposited in the Protein Data Bank. However, structural information on MTMR1 is not yet available. Structural studies of various MTMR proteins might be helpful in order to understand the detailed function of individual MTMRs at the molecular level. In order to improve our understanding of MTMR1 by comparing it with other MTMR structures, we initiated this study aimed at the structural determination of MTMR1. As the first step towards structural determination, we overexpressed and crystallized recombinant MTMR1. Diffraction data were then collected to 2.0 Å resolution using synchrotron radiation.

2. Materials and methods  

2.1. Macromolecule production  

The constructed MTMR1 gene (encoding amino-acid residues 95–607) was amplified using the polymerase chain reaction and inserted into modified pET-32 vector (Novagen). The active-site cysteine was then replaced with serine (C438S) using the QuikChange method. The resulting recombinant MTMR1 protein contains ten extra residues at its C-terminus (LEHHHHHHHH) for purification (Table 1 ▶). The recombinant protein was overexpressed in Escherichia coli strain Rosetta2 (DE3) cells (Novagen). The transformed cells were grown in Luria–Bertani medium containing 50 µg ml⁻¹ kanamycin at 310 K. Expression of recombinant MTMR1 was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) when the cells had reached an optical density (OD_600 nm) of 0.5. The cell cultures were induced with IPTG for 20 h at 291 K. The cells were then harvested by centrifugation at 8000g for 20 min at 277 K. The cell pellet was resuspended in ice-cold lysis buffer (20 mM Tris–HCl pH 8.0, 10 mM Na₂HPO₄, 300 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) and the cells were disrupted by sonication. The lysate was centrifuged at 13 000g for 1 h at 277 K. The supernatant was loaded by gravity onto a Glass Econo-Column (Bio-Rad) packed with Ni²⁺-chelated Ni–NTA agarose resin (Qiagen) which had previously been equilibrated with buffer A (20 mM Tris–HCl pH 8.0, 10 mM Na₂HPO₄, 300 mM NaCl) and was subsequently washed with buffer A containing 50 mM imidazole. The protein was eluted with buffer A containing 400 mM imidazole. Final purification of the protein was achieved by size-exclusion chromatography. The sample was injected onto a HiLoad 16/600 Superdex 200 pg (GE Healthcare) column that had been pre-equilibrated with buffer B (20 mM Tris–HCl pH 8.0, 300 mM NaCl, 10 mM Na₂HPO₄, 5 mM dithiothreitol). The purified protein was concentrated to 13.5 mg ml⁻¹ using an Amicon Ultra-15 ultrafiltration device (Millipore). The homogeneity of the purified protein was determined by SDS–PAGE under denaturing conditions using 12%(v/v) polyacrylamide gels. The protein concentration was determined by the Bradford method (Bradford, 1976 ▶) using bovine serum albumin as a standard. Dynamic light-scattering (DLS) experiments were performed on a DynaPro Titan instrument (Wyatt Technology) at 298 K and 20% laser power. Purified protein was identified by MALDI-TOF/TOF mass spectrometry. Analysis was performed using a 4700 Proteomics Analyzer (AB Sciex). The acquired MS/MS spectra were analyzed against the Swiss-Prot database using ProteinPilot v.5.0 (AB Sciex).

Table 1. Macromolecule-production information.

Source organism	Homo sapiens
DNA source	Open Biosystems (catalogue No. MHS1010-7295807)
Forward primer†	5-GGAATTCCATATGGAAGAGGCTCCACTTTT-3
Reverse primer‡	5-CCGCCGCTCGAGCCGTGGATTCCATCGTACATA-3
Expression vector	Modified pET-32
Expression host	E. coli Rosetta2 (DE3)
Complete amino-acid sequence of the construct produced§	MEEAPLFPGESIKAIVKDVMYICPFMGAVSGTLTVTDFKLYFKNVERDPHFILDVPLGVISRVEKIGAQSHGDNSCGIEIVCKDMRNLRLAYKQEEQSKLGIFENLNKHAFPLSNGQALFAFSYKEKFPINGWKVYDPVSEYKRQGLPNESWKISKINSNYEFCDTYPAIIVVPTSVKDDDLSKVAAFRAKGRVPVLSWIHPESQATITRCSQPLVGPNDKRCKEDEKYLQTIMDANAQSHKLIIFDARQNSVADTNKTKGGGYESESAYPNAELVFLEIHNIHVMRESLRKLKEIVYPSIDEARWLSNVDGTHWLEYIRMLLAGAVRIADKIESGKTSVVVHSSDGWDRTAQLTSLAMLMLDSYYRTIKGFETLVEKEWISFGHRFALRVGHGNDNHADADRSPIFLQFVDCVWQMTRQFPSAFEFNELFLITILDHLYSCLFGTFLCNCEQQRFKEDVYTKTISLWSYINSQLDEFSNPFFVNYENHVLYPVASLSHLELWVNYYVRWNPRLEHHHHHHHH

^†The NdeI site is underlined.

^‡The XhoI site is underlined.

^§The C438S mutation site and the ten-residue cloning artefact are underlined.

2.2. Crystallization  

The initial crystallization screening was performed using the ProPlex kit (Molecular Dimensions) by the sitting-drop vapour-diffusion method in 96-well plates at 277 K. The best crystals were grown in reservoir solution consisting of 100 mM Na HEPES pH 7.0, 15%(v/v) polyethylene glycol 20 000 (PEG 20K) and used for X-ray diffraction experiments (Table 2 ▶).

Table 2. Crystallization.

Method	Sitting-drop vapour diffusion
Plate type	MRC 96-well plate
Temperature (K)	277
Protein concentration (mgml1)	13.5
Buffer composition of protein solution	20mM TrisHCl pH 8.0, 300mM NaCl, 10mM Na2HPO4, 5mM dithiothreitol
Composition of reservoir solution	100mM Na HEPES pH 7.0, 15%(v/v) polyethylene glycol 20000
Volume (l) and ratio of drop	2 (1:1 ratio of protein and reservoir)
Volume of reservoir (l)	70

2.3. Data collection and processing  

Several cryoprotectants were tested for data collection under cryogenic conditions, including glycerol, ethylene glycol, polyethylene glycol 400 and (±)-2-methyl-2,4-pentanediol. These results confirmed glycerol to be the best cryoprotectant. The crystal was transferred into reservoir solution containing 25%(v/v) glycerol for data collection under cryogenic conditions before being flash-cooled in liquid nitrogen. X-ray diffraction data were collected using an ADSC Q315r detector on beamline 5C at Pohang Light Source (PLS), Republic of Korea. The crystal was rotated through a total angle of 360° with 1.0° oscillation per frame. The exposure time was 3 s per frame (Table 3 ▶). The raw data were processed and scaled using the HKL-2000 software package (Otwinowski & Minor, 1997 ▶). MOLREP from the CCP4 program package was used for molecular replacement (Vagin & Teplyakov, 2010 ▶; Winn et al., 2011 ▶).

Table 3. Data collection and processing.

Values in parentheses are for the outer shell.

Diffraction source	Beamline 5C, PLS
Wavelength ()	0.9796
Temperature (K)	100
Detector	ADSC Q315r
Crystal-to-detector distance (mm)	300
Rotation range per image ()	1
Total rotation range ()	360
Exposure time per image (s)	3
Space group	P1
Unit-cell parameters (, )	a = 67.219, b = 96.587, c = 97.581, = 87.597, = 86.072, = 77.327
Mosaicity ()	0.4
Resolution range ()	50.02.0 (2.032.00)
Total No. of reflections	343072
No. of unique reflections	136981
Completeness (%)	84.7 (63.2)
Multiplicity	2.5 (2.0)
I/(I)	18.9 (2.6)
R r.i.m. † (%)	8.3 (39.2)
Overall B factor from Wilson plot (2)	44.6

^†As this value was not available, it was estimated by multiplying the R _merge value by the factor [N/(N 1)]^1/2, where N is the data redundancy.

3. Results and discussion  

In this study, MTMR1 was overexpressed in E. coli in a soluble form, purified and crystallized for structural studies. The purification yield was about 14.6 mg purified protein per litre of E. coli culture. SDS–PAGE analysis of purified MTMR1 showed that the protein was highly purified and suitable for crystallization (Fig. 1 ▶). The elution profile of MTMR1 on a HiLoad 16/600 Superdex 200 prep-grade column (120 ml column volume) exhibited a large single peak at 87 ml elution volume (Fig. 1 ▶). The native molecular weight of the recombinant protein as estimated by dynamic light-scattering analysis was ∼69 kDa, with a polydispersity of 13.7%, suggesting that purified MTMR1 exists as monomers in solution owing to truncation of the coiled-coil domain at the C-terminus. When the purified protein was analysed by MALDI-TOF/TOF, it was identified as MTMR1 (Fig. 2 ▶). Crystals were formed under various crystallization conditions, but most of them were not suitable for X-ray diffraction data collection owing to poor diffraction quality. The best crystals were obtained using a reservoir solution consisting of 100 mM Na HEPES pH 7.0, 15%(v/v) PEG 20K. The crystals used in the X-ray diffraction experiments grew to maximum dimensions of approximately 0.2 × 0.2 × 0.1 mm within six weeks (Fig. 3 ▶). Although the best crystals still exhibited diffraction patterns with smeared spots in some frames, it was possible to collect diffraction data. X-ray diffraction data were collected to 2.0 Å resolution (Fig. 3 ▶). The native crystals belonged to space group P1, with unit-cell parameters a = 67.219, b = 96.587, c = 97.581 Å, α = 87.597, β = 86.072, γ = 77.327°. Assuming the presence of four molecules in the asymmetric unit, the calculated Matthews coefficient (V _M) was 2.61 ų Da⁻¹ and the corresponding solvent content was 52.9% (Matthews, 1968 ▶). Data-collection statistics are shown in Table 3 ▶. Molecular replacement using the MTMR2 structure (PDB entry 1lw3; Begley et al., 2003 ▶) as a search model was successful and allowed the identification of four protein molecules in the asymmetric unit. Although the diffraction data exhibited low completeness, the resulting electron-density map was interpretable. Further refinement of the model structure is in progress.

Figure 1.

(a) SDS–PAGE analysis of purified MTMR1. The left lane contains molecular-weight marker (labelled in kDa). (b) SEC elution profile of MTMR1.

Figure 2.

Identification of the purified protein by MALDI-TOF/TOF analysis.

Figure 3.

(a) Crystals of human MTMR1. (b) X-ray diffraction pattern of a human MTMR1 crystal.