Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Mrs2, a magnesium ion transporter from yeast inner mitochondrial membrane

Soluble N-terminal domain of yeast magnesium ion transporter Mrs2 was overexpressed in E. coli, purified, crystallized and X-ray diffraction data collected to 1.83 Å resolution.

Abstract

Mrs2 transporters are distantly related to the major bacterial Mg²⁺ transporter CorA and to Alr1, which is found in the plasma membranes of lower eukaryotes. Common features of all Mrs2 proteins are the presence of an N-terminal soluble domain followed by two adjacent transmembrane helices (TM1 and TM2) near the C-terminus and of the highly conserved F/Y-G-M-N sequence motif at the end of TM1. The inner mitochondrial domain of the Mrs2 from Saccharomyces cerevisae was overexpressed, purified and crystallized in two different crystal forms corresponding to an orthorhombic and a hexagonal space group. The crystals diffracted X-rays to 1.83 and 4.16 Å resolution, respectively. Matthews volume calculations suggested the presence of one molecule per asymmetric unit in the orthorhombic crystal form and of five or six molecules per asymmetric unit in the hexagonal crystal form. The phase problem was solved for the orthorhombic form by a single-wavelength anomalous dispersion experiment exploiting the sulfur anomalous signal.

1. Introduction

Mg²⁺ is the most abundant divalent cation in cells and organelles (Diwan, 1987 ▶). It plays important roles in stabilizing macromolecules and in binding to nucleotides and acts as a cofactor of many enzymes. By regulating the activities of ion channels and transporters, Mg²⁺ also influences cell volume and signalling processes (Ikari et al., 2008 ▶; Mobasheri et al., 1998 ▶).

Mrs2 transporters form the major mitochondrial Mg²⁺-uptake system in yeast, plants and mammals (Zsurka et al., 2001 ▶; Kolisek et al., 2003 ▶; Li et al., 2001 ▶; Schock et al., 2000 ▶) and are essential for mitochondrial biogenesis (Walker et al., 1982 ▶). Mrs2 transporters are distantly related to the major bacterial Mg²⁺ transporter CorA and to Alr1, which is located in the plasma membrane of lower eukaryotes. The amino-acid sequence identity between the N-terminal domains of CorA from Thermotoga maritima and Mrs2 from Saccharomyces cerevisae is 11%. The members of the Alr1 subfamily appear to be restricted to lower eukaryotes, where they form the major Mg²⁺-uptake system of the plasma membrane. Expression of Alr1 is essential for the growth of yeast cells, except when kept in media with nonphysiologically high Mg²⁺ concentrations (Graschopf et al., 2001 ▶). On the other hand, the cellular uptake of Mg²⁺ in mammals is mediated by proteins unrelated to the CorA–Mrs2–Alr1 superfamily that belong to the mammalian melastatin-related transient receptor potential (TRPM) family of cation channels (TRPM6 and TRPM7; Voets et al., 2004 ▶; Schmitz et al., 2005 ▶).

Common features of all of these proteins are the presence of two adjacent transmembrane helices (TM1 and TM2) near the C-­terminus and of the highly conserved F/Y-G-M-N sequence motif at the C-­terminus of TM1 (Bui et al., 1999 ▶; Gardner, 2003 ▶; Knoop et al., 2005 ▶; Lunin et al., 2006 ▶). The N-terminus is characterized by a large cytoplasmic domain which forms a funnel and has been shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state (Payandeh et al., 2008 ▶).

Despite low amino-acid sequence identity, secondary-structure features appear to be conserved in the N-terminal and transmembrane portions of all members of the CorA–Mrs2–Alr1 superfamily (Lunin et al., 2006 ▶), while they display notable differences in the region C-terminal to TM2 (Gardner, 2003 ▶). Moreover, some of these transporters can partially replace each other, which strongly supports the notion that they are orthologues (Bui et al., 1999 ▶; Lunin et al., 2006 ▶; Li et al., 2001 ▶; R. Schweyen, unpublished work).

Mrs2 is located in the inner mitochondrial membrane, while Alr1 is inserted into the cytoplasmic membrane. The orientation of these transporters in their cognate membranes is such that both the long sequence N-terminal to TM1 and the sequences C-terminal to TM2 are oriented towards the mitochondrial matrix and the cytoplasm in Mrs2 and Alr1, respectively. Only a short loop connecting the TM helices is oriented towards the space between the two mitochondrial membranes and outside in Mrs2 and Alr1, respectively (Baumann et al., 2002 ▶; Bui et al., 1999 ▶; Wachek et al., 2006 ▶). In their cognate membranes yeast Mrs2 and Alr1 have been shown to form homo-oligomers, while the bacterial transporter CorA was found to be in a pentameric state (Kolisek et al., 2003 ▶; Wachek et al., 2006 ▶; Warren et al., 2004 ▶; Weghuber et al., 2006 ▶).

Here, we report the crystallization and X-ray diffraction analysis of the N-terminal domain of Mrs2, the Mg²⁺ transporter from yeast inner mitochondrial membrane.

2. Materials and methods

2.1. Cloning and expression

The gene encoding the mitochondrial magnesium transporter Mrs2 (YOR334W) from S. cerevisae was used to amplify the mitochondrial matrix domain of Mrs2 (Mrs2_16–276; M _r = 30 124) by polymerase chain reaction (PCR) using Taq DNA polymerase with the forward primer 5′-CTTTATTTTCAGGGCGCCATGGGCAAACAGTTACTATCGTTGAAGCCCATT-3′ and the reverse primer 5′-GTGCTCGAGTGCGGCCGCTTATAAGGAATTTCTATTTGCGTCCAATATGAT-3′, which were designed based on the nucleotide sequence of the Mrs2 gene. The PCR product was digested with NcoI and NotI (the recognition sites are shown in bold in the primers used) and cloned into the vector pETM-11 (EMBL Hamburg) with a tobacco etch virus (TEV) cleavable N-terminal His₆ tag MKHHHHHHPMSDYDIPT­TENLYFQGA. The DNA insert cloned in pETM-11 was sequenced to confirm the fidelity of DNA amplification.

The recombinant protein was overexpressed in BL-21 Star (DE3) Escherichia coli transformants grown in LB medium containing kanamycin (0.025 mg ml⁻¹) at 310 K with shaking at 200 rev min⁻¹; the cells were induced at an OD₆₀₀ of 0.6 with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and allowed to grow to a final OD₆₀₀ of 2.3 at 294 K. The cells were harvested by centrifugation (6000g, 30 min), resuspended in sonication buffer (50 mM Tris–HCl pH 8.0, 500 mM NaCl) supplemented with 1 mM phenylmethanesulfonylfluoride (PMSF), 5 mM β-mercaptoethanol (BME), 20 mM imidazole and 5% glycerol, and lysed by sonication.

2.2. Purification

After sonication of the cells, the crude extract was centrifuged at 50 000g for 30 min and the supernatant was loaded onto a 5 ml Ni–NTA agarose column (Qiagen) pre-equilibrated in buffer A (50 mM Tris–HCl pH 8.0, 500 mM NaCl, 1 mM PMSF, 5 mM BME, 20 mM imidazole and 5% glycerol). The column was extensively washed with buffer A containing 20 mM imidazole. The protein was eluted with a linear gradient of buffer B containing 500 mM imidazole. The N-­terminal His₆ tag was cleaved using TEV (protease:protein mass ratio 1:70) for 12 h during dialysis against 50 mM Tris–HCl pH 8.0 containing 300 mM NaCl, 1 mM dithiothreitol (DTT) and 1 mM PMSF. The cleaved N-terminus and TEV were separated from the protein after TEV cleavage by again applying the reaction mixture onto the Ni–NTA column. The flowthrough was pooled and loaded onto a Resource Q column (6 ml; GE Healthcare) equilibrated with buffer A consisting of 50 mM Tris–HCl pH 8.0, 20 mM NaCl, 5 mM BME and 1 mM PMSF and the protein was eluted using a gradient of 20–1000 mM NaCl in 50 mM Tris–HCl pH 8.0, 5 mM BME and 1 mM PMSF. The protein eluted from the Resource Q column in a single peak at 280 mM NaCl. Further purification was carried out on a HiLoad 26/60 Superdex 200 (GE Healthcare) gel-filtration column in 50 mM Tris–HCl pH 8.0 and 300 mM NaCl. The protein eluted in a single peak at an elution volume of 218 ml, corresponding to a molecular weight of 30 kDa, which is compatible with the monomer of Mrs2_16–276. The purified protein was concentrated by reapplying it onto a Resource Q column (6 ml; GE Healthcare) as described above; it was concentrated to about 3 mg ml⁻¹ for crystallization screening. These procedures reproducibly yielded 6 mg protein from 1 l of bacterial culture.

The purity of the protein solution used in the crystallization experiments was checked by SDS–PAGE analysis and showed a single band with an apparent molecular weight of about 30 kDa (Fig. 1 ▶ a).

Figure 1.

SDS–PAGE gels of TEV cleavage of Mrs2_16–276 and of dissolved Mrs2_16–276 crystals (form A). (a) Lane 1, before His₆-tag cleavage; lane 2, after His₆-tag cleavage with TEV; lane S, molecular-weight standards (kDa). (b) Lane C, control; lane Cr, dissolved crystals; lane S, molecular-weight standards (kDa).

Dynamic light scattering was used to assess the monodispersity of the protein solution: a monomodal distribution with a polydispersity of 5% was observed and the gyration radius was estimated to be 4.2 nm, suggesting that the protein solution was homogenous and monomeric. Circular-dichroism spectroscopy in the far-ultraviolet wavelength range showed that the protein was rich in α-helical content.

2.3. Crystallization

Initial screening for crystallization conditions was performed using Crystal Screens 1 and 2, PEG/Ion Screen, Index, Natrix, SaltRX, Cryo 1 and 2, JCSG, PACT and MembFac kits from Hampton Research. A nanodrop crystallization robot (Phoenix RE, Matrix Technologies) was employed for screening using the sitting-drop vapour-diffusion method at 295 K, mixing equal volumes (0.2 µl) of protein solution (3 mg ml⁻¹ in anion-exchange buffer) and reservoir solution. Crystals of Mrs2_16–276 were initially obtained from Cryo 1 and 2 condition No. 59, consisting of 0.1 M sodium/potassium phosphate pH 6.2, 25%(v/v) 1,2-propanediol, 10%(v/v) glycerol, and condition No. 22, composed of 0.1 M sodium/potassium phosphate pH 6.2, 40%(v/v) ethylene glycol. For optimization of crystallization conditions, 1 µl protein solution (3 mg ml⁻¹ in anion-exchange buffer) was mixed with 1 µl mother liquor for vapour-diffusion experiments against 0.5 ml reservoir solution. After further optimization of the crystallization conditions, the best crystals were obtained using 22%(v/v) ethylene glycol, 56 mM sodium/potassium phosphate pH 6.3. The crystals (form A) appeared overnight and grew to maximum dimensions of about 0.5 × 0.2 × 0.2 mm within 2–3 d (Fig. 2 ▶ a). Small crystals of Mrs2_16–276 (form B) were also obtained in the presence of 1.7 M NaCl, 70 mM imidazole pH 7.8 at 295 K (Fig. 2 ▶ b). However, these crystals did not diffract to high resolution.

Figure 2.

The two different forms of Mrs2_16–276 crystals. (a) Crystals of Mrs2_16–276 grown in 22%(v/v) ethylene glycol, 56 mM sodium/potassium phosphate pH 6.3 (form A). (b) Crystals of Mrs2_16–276 grown in 1.7 M NaCl, 70 mM imidazole pH 7.8 (form B).

The crystals (form A) were dissolved in deionized water at room temperature. SDS–PAGE was performed on 12.5% polyacrylamide. The protein bands of dissolved crystals were identified by comparing their mobility with those of protein standard molecular-weight markers and purified Mrs2_16–276 (Fig. 1 ▶ b).

2.4. X-ray diffraction

All Mrs2_16–276 X-ray diffraction data sets were collected at 100 K in a cold nitrogen stream using either an in-house Bruker Microstar rotating-anode generator equipped with a Platinum 135 CCD detector and a 300 µm collimator or the ID14-1 beamline at ESRF equipped with an ADSC Q210 CCD detector, using a beam size of 100 × 100 µm. Prior to cryocooling in a nitrogen stream, the form A crystals of Mrs2_16–276 were transferred into a cryoprotectant solution containing 30%(v/v) ethylene glycol, 56 mM sodium/potassium phosphate pH 6.3, while the form B crystals were transferred into a cryoprotectant solution containing 19% glycerol, 1.7 M NaCl, 70 mM imidazole pH 7.8. The form A crystals of Mrs2_16–276 diffracted to 1.83 Å resolution on an in-house source (Fig. 3 ▶ a) and belonged to space group P2₁2₁2₁ (unit-cell parameters a = 54.66, b = 67.70, c = 85.30 Å), with the asymmetric unit containing one molecule. This is consistent with a Matthews coefficient of 2.32 ų Da⁻¹ and a solvent content of 47.0%. The form B crystals of Mrs2_16–276 diffracted to 4.16 Å resolution at ESRF (Fig. 3 ▶ b) and belonged to space group P6₂22 or P6₄22 (unit-cell parameters a = b = 230.00, c = 114.47 Å), with the asymmetric unit being likely to contain five or six molecules, corresponding to a Matthews coefficient of 2.84 ų Da⁻¹ and a solvent content of 56.7%.

Figure 3.

Diffraction patterns of Mrs2_16–276 crystals. (a) Diffraction pattern of form A Mrs2_16–276 crystals. The resolution is ∼1.8 Å at the edge. (b) Diffraction pattern of form B Mrs2_16–­276 crystals. The resolution is ∼3.4 Å at the edge.

The data-collection statistics are summarized in Table 1 ▶. Crystallo­graphic data collected in-house were processed (integrated and scaled) with the PROTEUM2 software suite (Bruker AXS Inc.), while the synchrotron data sets were processed using XDS (Kabsch, 2010 ▶).

Table 1. Data-collection statistics for the N-terminal domain of Mrs2.

Values in parentheses are for the outermost resolution shell. No σ(I) cutoff was used in the data-integration process.

	Form A (home source)	Form B (ESRF ID14-1)
Wavelength (Å)	1.54178	0.933
Temperature (K)	100	100
Space group	P212121	P6422 or P6222
Unit-cell parameters (Å, °)	a = 54.66, b = 67.70, c = 85.30, α = β = γ = 90	a = b = 230.00, c = 114.47, α = β = 90, γ = 120
Resolution (Å)	36.89–1.83 (1.90–1.83)	50–3.60 (4.25–4.16)
Measured reflections	3977702	365747
Unique reflections	48844	25957
Completeness (%)	99 (92)	96 (90)
Redundancy	80 (13)	27 (28)
Anomalous completeness (%)	92.5 (83.0)	N/A
No. of subunits in asymmetric unit	1	5 or 6
Phasing power	0.311 (0.069)	N/A
Figure of merit (centric/acentric)	0.08586/0.21770	N/A
Rp.i.m.† (%)	0.80 (23.0)	N/A
Rmerge‡ (%)	8.5 (88.4)	9.6 (65.9)
Rmeas§ (%)	N/A	9.8 (67.0)
〈I/σ(I)〉	40.2 (1.9)	31.7 (6.5)

^† R _p.i.m. = , where I_i(hkl) and 〈I(hkl)〉 are the ith and the mean measurements of the intensity of reflection hkl and N is the redundancy.

^‡ R _merge = .

^§ R _meas = .

2.5. Solution of the phase problem

The structure could not be solved by molecular replacement using the known CorA structure as a search model (Lunin et al., 2006 ▶), but was instead solved by the single-wavelength anomalous dispersion method (SAD) using the Mrs2 native S atoms for phasing. A highly redundant (average multiplicity of 80) data set with high anomalous completeness (92.5%; 83.0% in the outermost shell) was obtained from a form A crystal using the in-house X-ray source with a 1.54 Å wavelength (Table 1 ▶). The diffraction data were cut to 2.5 Å resolution to determine the sulfur substructure using SHELXD (Sheldrick, 2008 ▶). All 11 S-atom sites were found, corresponding to the five cysteine and six methionine residues present in the protein. Subsequent heavy-atom refinement and density modification was per­formed using autoSHARP (Vonrhein et al., 2007 ▶). The sulfur SAD phases obtained from autoSHARP produced an electron-density map which was traced using ARP/wARP (Morris et al., 2003 ▶; Joosten et al., 2008 ▶) and automatically fitted 243 of the 261 amino-acid residues in three different chains (R _work and R _free of 0.21 and 0.30, respectively) into the density. Structure determination is currently in progress using the diffraction data extending to 1.83 Å resolution.