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Molecular characterization of methanogenic N5-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase
Biochimica et Biophysica Acta 1858 (2016) 2140–2144
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Molecular characterization of methanogenic N5-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase Vikrant Upadhyay a, Katharina Ceh a, Franz Tumulka b, Rupert Abele b, Jan Hoffmann c, Julian Langer a, Seigo Shima d,e,⁎, Ulrich Ermler a,⁎ a
Max-Planck-Institut für Biophysik, Max-von-Laue-Straße 3, D-60438 Frankfurt am Main, Germany Goethe-Universität Frankfurt am Main, Institut für Biochemie, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany Institut für Physikalische und Theoretische Chemie, Goethe-Universität Frankfurt am Main, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany d Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, Germany e PRESTO, Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Saitama 332-0012, Japan b c
a r t i c l e
i n f o
Article history: Received 4 May 2016 Accepted 15 June 2016 Available online 21 June 2016 Keywords: Methanogenesis N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase Membrane protein complex SEC-MALS LILBID-MS Dimethyl maleic anhydride
a b s t r a c t Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyltetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS. © 2016 Published by Elsevier B.V.
1. Introduction Methanogenic archaea annually produce ca. 1 billion tons of methane - an important greenhouse gas and energy resource - in anoxic habitats, such as water sediments, rice fields, hydrothermal vent peripheries as well as intestinal tracts of animals [1]. Acetate, CO2, methanol and few other one-carbon substrates are covalently bound in different oxidation states to the three one-carbon carriers methanofuran, tetrahydromethanopterin (H4MPT) and coenzyme M (HS-CoM) and reduced to methane by various electron donors [1]. The only ion-gradient forming process universally applied in the energy metabolism of methanogenic archaea is accomplished by N5-methyl-H4MPT: HS-CoM methyltransferase (Mtr). This multi-subunit membrane protein complex catalyzes the transfer of the methyl group from methyl-H4MPT to
⁎ Corresponding authors. E-mail addresses: [email protected] (S. Shima), [email protected] (U. Ermler).
http://dx.doi.org/10.1016/j.bbamem.2016.06.011 0005-2736/© 2016 Published by Elsevier B.V.
coenzyme M coupled with the vectorial translocation of Na+ across the cytoplasmic membrane [2–4]. Naþ
CH3 ‐H4 MPT þ H‐S‐CoM → CH3 ‐S‐CoM 0 þ H4 MPT ΔG ° ¼ −30 kJ=mol The Mtr complex was first isolated ca. 20 years ago from Methanothermobacter marburgensis (formerly known as Methanobacterium thermoautotrophicum strain Marburg) [5] and Methanosarcina mazei strain Gö1 [6]. The membrane fraction of M. marburgensis was extracted with 2.5% dodecyl-β- D -maltoside and the solubilized Mtr complex purified via chromatography on DEAE sepharose, Q sepharose, Superose 6 and Mono Q. The membrane-spanning complex is composed of eight subunits with molecular masses (Mr) of 25.6 kDa for MtrA, 10.7 kDa for MtrB, 27.1 kDa for MtrC, 22.8 kDa for MtrD, 31.2 kDa for MtrE, 7.3 kDa for MtrF, 9.5 kDa for MtrG and 33.5 kDa for MtrH of M. marburgensis. The apparent Mr of the whole enzyme complex was determined to be 670 kDa by size exclusion chromatography [7]. This finding led to the conclusion
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that the Mtr complex is a tetramer of heterooctamers Mtr(ABCEDFGH)4. Primary structure analysis indicates that MtrC, MtrD and MtrE are integral membrane proteins with seven, six and six membrane-spanning helices, respectively. MtrE potentially hosts a highly conserved zinc binding motif Asp-X-Glu-X22-His-X9-Glu [8] which is characteristic for all known Zn2+ binding enzymes catalyzing thiol alkylation [9,10] and an invariant aspartate in the membrane matrix which has been already reported as essential residue in several Na+ or H+ pumping protein complexes [4,11,12]. MtrA, MtrB, MtrF and MtrG only contain one transmembrane helix anchor. The soluble domain of MtrA is directed to the cytoplasm and contains an 5-hydroxy-benzimidazolyl-cobamide cofactor [5], also called factor III, which is a homologue to vitamin B12 (5, 6-dimethyl-benzimidazolyl-cobamide). Active Mtr requires cobalt of 5-hydroxy-benzimidazolyl-cobamide in the oxidation state I. MtrH is the only subunit of the Mtr complex without a membrane-spanning region and can be easily lost during cell lysis and solubilization [5]. Based on the obtained molecular and kinetic data, the reaction proceeds by a methyl transfer of methyl-H 4MPT bound to MtrH to cob(I)alamin bound to MtrA [13] thereby forming methylcob(III)alamin containing the methyl group and a histidine as axial ligands. Methyl-cob(III)alamin transfers its methyl group to coenzyme M forming methyl-coenzyme M and returns to the cob(I)alamin state. The latter reactions were shown to be dependent on Na+ [14,15] and might be therefore coupled with its vectorial translocation. However, for unravelling its complicated catalytic mechanism, larger amounts of a homogeneous protein sample and more detailed structural information are required. As a first step we developed a procedure to isolate a highly pure and homogeneous MtrA-G complex from M. marburgensis and characterized the prepared multisubunit membrane complex in terms of the absolute molecular mass and the oligomeric state. 2. Materials and methods 2.1. Materials M. marburgensis was obtained from the Deutsche Sammlung für Microorganismen (Braunschweig, Germany). The archaeon was grown as described previously [16]. Dodecyl β-D-maltoside (DDM) and dodecyl tricosaoxyethylene glycol ether (Brij-35) was purchased from Anatrace and dimethyl maleic anhydride (DMMA) from SigmaAldrich. All the chromatographic columns and column materials were acquired from GE Healthcare Lifesciences.
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final protein concentration of 200 μg/ml followed by adding solid DMMA (2 mg/ml) to the suspension. The pH of the suspension was kept at 8.0 by adding 1 M NaOH solution while continuously stirring. When the pH became constant and almost all DMMA was dissolved, the membrane was pelleted by centrifugation at 100,000 × g for 1.5 h. The membranes were then resuspended in 14 ml solubilization buffer (50 mM MOPS-NaOH pH 7.0, 10 mM MgCl2, 2 mM dithiothreitol and 2.5% w/v DDM) and incubated at 4 °C for overnight. The nonsolubilized membrane was pelleted by centrifugation at 100,000 × g for 1.5 h. The supernatant containing the solubilized MtrA-G complex was loaded to a DEAE-sepharose column (10 ml) previously equilibrated with purification buffer (50 mM MOPS-NaOH pH 7.0, 100 mM NaCl, 10 mM MgCl 2, 2 mM dithiothreitol) and 0.1% w/v DDM. The proteins were eluted with a linear 150 ml gradient of 0.1–1.0 M NaCl. The fractions containing the MtrA-G complex were selected for SDS-PAGE analysis. Size exclusion chromatography on a Superose-6 column was finally performed with the purification buffer supplemented with 0.1% w/v DDM and 10% glycerol for removing any remaining impurities and soluble aggregates and to evaluate the homogeneity of the purified complex. Detergent exchange to Brij-35 was achieved by washing and eluting MtrA-G with purification buffer containing 0.2% Brij-35 on the anion exchange DEAE-sepharose column. Purification was completed with size exclusion chromatography.
2.3. Laser induced liquid beam ion desorption mass spectrometry MtrA-G was transferred into 20 mM Tris, pH 7.0, 2 mM dithiothreitol and 0.1% w/v DDM with ultrafiltration prior to LILBID-MS analysis [17]. The spectra were recorded twice. The ion source was a commercial droplet dispenser (Microdrop), which injects microdroplets of 50 μm radius and 65 pl volume from pressure reduction apertures into high vacuum. The droplets were irradiated one-by-one by high intensity mid-IR laser pulses (wavelength ca. 3 μm) from a home-built Nd:Yag pumped LiNbO3 optical parametric oscillator. The mass analysis was performed by a home-built time of flight mass spectrometer with a Wiley-McLaren-type acceleration region and an ion reflector (Reflectron). The ions with large m/z (up to ~ 10 6) were detected by a home-built Daly-type high mass detector. Only 200 droplets were sampled for each mass spectrum. 10 μl of sample (ca. 9.0 mg/ml) was used in each analysis.
2.2. Protein production
2.4. Multi-angle light scattering
2.2.1. Protein isolation The MtrA-G complex was isolated from the membrane fraction of M. marburgensis cell as described earlier [16] with some modifications. All the steps were performed at 4 °C, if not stated otherwise. 10 g of frozen cells were thawed and resuspended in 50 ml lysis buffer (50 mM MOPS-NaOH pH 7.0, 10 mM MgCl2 and 2 mM dithiothreitol) containing 5 mg bovine pancreatic DNase I (Roche). Cells were disrupted by passing them through a microfluidizer (Microfluidics) for 4 times at 16,000 psi pressure. The cell lysates were centrifuged twice at 10,000 ×g for 30 min to remove cell debris. The membrane was pelleted from the supernatant by centrifugation at 100,000 ×g for 1.5 h, resuspended in the lysis buffer and pelleted again. After resuspending in the lysis buffer to the final volume of 5 ml, the membrane pellet was either used immediately for MtrA-G preparation or shock frozen in liquid nitrogen before storing it at −80 °C till further use.
Multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) was performed on a TSKgel G4000SWxl column (Tosoh Biosciences) at a flow rate of 0.4 ml/min on a Jasco HPLC unit (Jasco Labor und Datentechnik) connected to a light scattering detector measuring at three angles (miniDAWN TREOS, Wyatt Technology) and a refractive index detector (Optilab T-rEX, Wyatt Technology). The column was equilibrated for at least 16 h with purification buffer supplemented with 0.1% w/v DDM and 10% glycerol. The buffer was filtered through 0.1 μm pore size VVLP filters (Millipore) before applying to the column. 200 μl of protein samples (ca. 1.6 mg/ml) were separated on the column. Data analysis was accomplished using the ASTRA software package 5.3.4.13 (Wyatt Technology) and data fitting was done with a Zimm's model [18]. The refractive index increment dn/dc values of the protein and of DDM were taken as 0.185 ml/g and 0.133 ml/g respectively from the literature. The extinction coefficients of individual Mtr subunits at 280 nm were calculated using ProtParam server [19] whereas that of corrinoid prosthetic group was taken from literature [20]. The sum of the extinction coefficients of individual subunits and cobalamin (866 ml/g cm−1) was used in the data analysis.
2.2.2. Protein preparation of the MtrA-G complex after DMMA treatment The membranes from 10 g cells were resuspended at room temperature in extraction buffer (50 mM HEPES-NaOH pH 8.0, 10 mM MgCl2, 150 mM NaCl and 2 mM dithiothreitol) to adjust the
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3. Results and discussion 3.1. Preparation of a homogeneous MtrA-G complex The aerobic purification of the MtrA-H or MtrA-G complexes from the membrane fraction of M. marburgensis started from a former protocol developed for an isolation under strictly anoxic conditions. [5] To preserve the catalytic activity of the Mtr complex cobalt of 5hydroxybenzimidazolyl-cobamide has to be kept in a highly O2 sensitive oxidation state I. Despite its relative purity and integrity, the obtained Mtr complex was not sufficiently homogeneous for serious structural studies. The major problem was the loss of substantial amounts of MtrH from the membrane fraction essentially during cell lysis and membrane protein solubilization. Although the separation of MtrA-G from MtrA-H and MtrH was, in principle, possible during chromatographic purification on DEAE-sepharose [13] the amount of pure MtrA-G was very small. Extensive attempts were performed either to completely remove the non-covalently attached MtrH or to quantitatively maintain its association. However, MtrH was lost during the cell rupture independent of the used techniques although microfluidization kept the integrity of the MtrA-H complex to a higher extent. Dissociated MtrH was clearly detectable in the cytoplasmic fraction of the lysed M. marburgensis cells by SDS-PAGE and Western Blot analysis using monoclonal antibody specific to MtrH from M. marburgensis (Fig. S1). For obtaining a pure MtrA-G complex or to clearly separate MtrA-G and MtrA-H with a sufficient yield, purification was performed by exchanging the detergent DDM (CHAPS, Triton-X100, β-octylglucoside, LDAO, cholate), by variations in salt concentration (0.1–2.0 M NaCl) and pH (4.0–9.0) and after a high temperature incubation step (60 °C). Unfortunately, all these attempts failed. The breakthrough was achieved by using mild membrane perturbing agents such as low concentrations of urea or guanidine hydrochloride, alkaline pH, chaotropic salts and dicarboxylic anhydrides which were applied in the membrane-integrated state before solubilization. The most suitable agents was finally the dicarboxylic anhydride DMMA which selectively modifies at pH 8.0–9.0 free amino groups of lysines solely in water-accessible regions resulting in the disturbance of ionic interactions between the membrane-embedded and associated soluble subunits [21]. DMMA almost quantitatively detaches the water-accessible subunits of Mtr but also of other membrane proteins (facilitated further purification) and thereby does not disrupt the MtrA-G complex. An analogous result was reported for several other membrane proteins with associated soluble subunits [22–24].
Subsequent isolation steps were carried out at pH 7.0 where the modified lysine-DMMA adducts are unstable and revert back to the free amino groups. Notably, MtrH released after DMMA treatment remains intact and could be purified from the soluble fraction by ion exchange and size exclusion chromatography (Fig. S1). After solubilization of MtrA-G with DDM the enzyme complex was purified by anion exchange followed by size exclusion chromatography, instead of four steps as described in the original protocol. The MtrA-G complex was eluted in a nearly homogeneous form after passing the final size exclusion column (Fig. 1A). The yield of purified MtrA-G complex was ca. 1 mg per 10 g of wet cell mass as determined by standard Bradford assay. The Coomassie stained gel after SDS-PAGE again showed the presence of all MtrA-G subunits and the almost complete removal of MtrH (Fig. 1B). The presence of the individual Mtr subunits was ascertained by peptide mass fingerprinting analysis using for digestion either trypsin or chymotrypsin (Table S1 and S2). The success of the used method becomes obvious when comparing the MtrH content with and without treatment with DMMA (Fig. 1B and Fig. S2). Although the quality of the purification was sufficient for the subsequent molecular mass determination we continuously worked on a further improvement of the homogeneity of the protein complex for further structural studies and found out that that a detergent exchange from DDM to Brij-35 provides a more symmetric gel filtration profile (Fig. S3). 3.2. Molecular mass determination The apparent Mr of MtrA-H in the presence of 0.1% DDM was determined to ca. 670 kDa (similar to thyroglobulin) on the basis of the elution time of size exclusion chromatographic runs [5]. This value suggested a tetrameric oligomeric state because the total Mr of the individual subunits of Mtr was calculated to be 155 kDa based on a SDS-PAGE profile. Subunit MtrB with a Mr of 12.5 kDa was originally not identified as part of the Mtr complex but its consideration does not change the result [5,7]. Size-exclusion based Mr determination is fairly reliable for soluble, sphere-shaped and non-aggregating proteins but for solubilized membrane proteins rather the Mr of the protein/detergent complex than that of the protein is obtained. The amount of the bound detergent is normally unknown [25]. Methods as LILBID-MS (laser induced liquid bead ion desorption mass spectrometry) [17], SEC-MALS (size exclusion chromatography-multiple angle light scattering) [26] and sedimentation equilibrium analytical ultracentrifugation [27] provide a more reliable Mr determination as their results are less shape- and detergent
Fig. 1. Purification of the MtrA-G complex. SEC profile (A) and Coomassie stained SDS-PAGE gel (B) of the purified complex. MtrA-G in DDM was eluted at ca. 1.39 ml from a 2.4 ml Superose-6 column. Lane 1: Molecular mass marker, lane 2: Purified MtrA-G sample. Bands above 32.5 kDa in size were higher oligomers of various subcomplexes of Mtr subunits as confirmed with peptide mass-fingerprinting.
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biased. Therefore, we determined the Mr of the homogeneous MtrA-G complex in the presence of 0.1% DDM by LILBID-MS and SEC-MALS. LILBID mass spectra were recorded with two different intensities of the mid-IR laser resulting in different degrees of dissociation. In the soft detection mode at very low laser intensity (Fig. 2A) the seven prominent m/z signals carrying 1 to 7 negative charges corresponded to a Mr of ca. 430 kDa. Although LILBID-MS can resolve mass differences of ca.
Fig. 2. LILBID mass spectra of MtrA-G at low (A) and high intensity (B and C) IR laser. Both experiments were performed two times with the samples from different preparations. An Mtr species with a Mr of ca. 430 kDa was dominant in the spectrum recorded at low intensity IR laser. At high intensity IR the Mtr complex dissociated into several subcomplex species and individual subunits. The Mr of the MtrA-G (sub)complexes are taken from the left side of the peak position. The size of the shoulder of the peaks indicates a variable content of attached small molecule dominated by DDM micelles. Letters A-H in (C) are abbreviations for MtrA-H.
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100 Da, non-covalently bound smaller molecules such as cobalamin, lipids, detergents, solvents (buffer, water, metal ions) and eventually post-translational modifications substantially broaden the peak. Therefore, the Mr is read out at the external left side of the peak containing the protein fraction and firmly attached small molecules [28]. The Mr is ca. 60–80 kDa higher at the external right side of the peak essentially due to the associated detergent micelle. A second signal of the MtrA-G complex with an Mr of 504 kDa was based on three m/z peaks with a width of ca. 60–70 kDa. Under harsher conditions (high intensity IR laser pulses, Fig. 2B + C) the MtrA-G complex disintegrated into several subcomplexes and individual subunits. Broad peaks are detectable at ca. 144 kDa, 107 kDa, 82 kDa and 73 kDa. More narrow but significantly high peaks were further visible at ca. 59 kDa, 54.5 kDa and 50 kDa. The complete decomposition and the presence of all subunits could be clearly demonstrated (Fig. 2C) at harsher conditions thereby confirming the results of peptide mass fingerprinting analysis (Table S1 and S2). SEC-MALS analysis yielded a weight averaged molecular mass (Mw) for the MtrA-G complex with and without the bound detergent of 578 and 428 kDa, respectively (Fig. 3) calculated on the basis of the threedetector method using the ASTRA software. The number averaged molecular mass (Mn) of the MtrA-G complex was very close to that of Mw, being 575 kDa and 428 kDa for the MtrA-G complex with and without bound detergent, respectively. Therefore, the polydispersity index (Mw/Mn) was close to unity indicating a high monodispersity of the protein sample. This finding is in line with the narrow elution peak of SEC (Fig. 3). Both, SEC-MALS and LILBID data recorded with a soft IR-laser resulted in a dominant first species with a Mr of the MtrA-G complex of ca. 430 kDa. Considering a genome sequence-based Mr of 134.2 kDa for the MtrA-G protomer, a trimeric oligomerization of the Mtr(A-G) complex appears to be most likely under the prerequisite that all subunits are present in a 1:1 stoichiometry. The discrepancy between the theoretical and measured Mr of ca. 27 kDa (430 kDa − 3 × 134.2 kDa) are most likely due to firmly bound small molecules composed of cobalamin, structural lipids, solvents and DDM. The value of ca. 8–10 kDa of small structural molecules per 100 kDa membrane protein is in line with the findings in AcrB [28] and virtually in complex I [29] but differ from those of ATPases [30,31].
Fig. 3. Molecular mass analysis of MtrA-G complex with SEC-MALS. MtrA-G was subjected to SEC-MALS analysis for the determination of its absolute Mr without detergent. The experiment was performed two times. The left axis represents Mw. Mn was calculated with the ASTRA software. The Mw and Mn of the protein fraction of the proteindetergent complex were calculated to 428 kDa giving the polydispersity index close to unity. The Mr of the protein-detergent complex, the protein and the detergent are marked with a black, red and blue line.
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A second species present in small amounts has a Mr of 504 kDa and 575 kDa based on LILBID-MS and SEC-MALS experiments, respectively. We primarily interpreted the excess mass as detergent micelles bound differently strong to the enzyme although the presence of minor amounts of MtrH has also been considered. When integrating the small structural molecules the Mr ratio protein/detergent of MtrA-G obtained by LILBID-MS is 5.8 (430 kDa/504–430 kDa) and by SEC-MALS 2.9 (430 kDa/580–430 kDa). Notably, the apparent Mr of 670 kDa reported for the MtrA-H-detergent complex [7] is also compatible with a trimeric oligomerization state. The Mr ratio protein/detergent is 3.0 (534 kDa/670–534 kDa) with the Mr of the MtrA-H trimer (3 ∗ 178 kDa) being composed of the mass derived from the gene sequence (168 kDa) and from small structural molecules (estimated to ca. 10 kDa). The application of harsher IR laser conditions resulted in the formation of subcomplexes (Fig. 2B). The peak at 144 kDa can be assigned to MtrA-G indicating that the protomer assembles to a compact, fairly interconnected unit, which less strongly interacts with the other protomers. Moreover, the discrepancy between measured and theoretical Mr (134.3 kDa) of ca. 10 kDa corresponds to the value for the entire (MtrA-G)3 complex per protomer. This extra mass is attributed to firmly attached small structural molecules and not as one of the small subunits resulting in a stoichiometry of the subunits MtrA-G of 1:1. The broad peak at ca. 107 kDa might correspond to either the MtrBCDEFG (108.7 kDa) protomer without the partly soluble MtrA or less likely the MtrABDEFG (107.1 kDa) subcomplex missing the membrane subunit MtrC. The MtrBCDEFG assignment is in line with the peak at ca. 82 kDa assigned as the MtrCDE (81 kDa) membrane core complex. Their significant peak width still indicates a considerable amount of attached small molecules. More narrow peaks at ca. 73, 59, 54.5 and 50 kDa can be tentatively assigned to MtrAEFG (73.7 kDa), MtrCDG (59.4), MtrBCFG (54.7 kDa) and MtrCD (49.9 kDa), respectively (Fig. 2B). Although the number and the composition of the detected subcomplexes do not convey a sufficiently complete picture about the subunit organization within the MtrA-G protomer, we tentatively constructed a preliminary schematic model (Fig. S4). Other techniques such as crosslinking and subsequent protease digestion peptide mass fingerprinting experiments as well as detailed structural methods are necessary for substantiation. The highly pure and homogeneous MtrAG complex provides a serious platform for these studies. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements We thank Rudolf K. Thauer and Hartmut Michel for continuous support. V.U. is grateful to the International Max Planck Research School for funding. We are grateful to Volker Müller for providing us with antibodies against ATP synthases from various methanogens. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamem.2016.06.011. References [1] R.K. Thauer, Biochemistry of methanogenesis: a tribute to Marjory Stephenson, Microbiology 144 (1998) 2377–2406. [2] B. Becher, V. Müller, G. Gottschalk, N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase of Methanosarcina strain Gö1 is an Na+-translocating membrane protein, J. Bacteriol. 174 (1992) 7656–7660.
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Pos, Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS, Biochim. Biophys. Acta Biomembr. 1808 (2011) 2189–2196. [29] N. Morgner, V. Zickermann, S. Kerscher, I. Wittig, A. Abdrakhmanova, H.-D. Barth, B. Brutschy, U. Brandt, Subunit mass fingerprinting of mitochondrial complex I, Biochim. Biophys. Acta Bioenerg. 1777 (2008) 1384–1391. [30] J. Hoffmann, L. Sokolova, L. Preiss, D.B. Hicks, T.A. Krulwich, N. Morgner, I. Wittig, H. Schägger, T. Meier, B. Brutschy, ATP synthases: cellular nanomotors characterized by LILBID mass spectrometry, Phys. Chem. Chem. Phys. 12 (2010) 13375–13382. [31] J. Vonck, K.Y. Pisa, N. Morgner, B. Brutschy, V. Müller, Three-dimensional structure of A1A0 ATP synthase from the hyperthermophilic archaeon Pyrococcus furiosus by electron microscopy, J. Biol. Chem. 284 (2009) 10110–10119.
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem
Molecular characterization of methanogenic N5-methyl-tetrahydromethanopterin: Coenzyme M methyltransferase Vikrant Upadhyay a, Katharina Ceh a, Franz Tumulka b, Rupert Abele b, Jan Hoffmann c, Julian Langer a, Seigo Shima d,e,⁎, Ulrich Ermler a,⁎ a
Max-Planck-Institut für Biophysik, Max-von-Laue-Straße 3, D-60438 Frankfurt am Main, Germany Goethe-Universität Frankfurt am Main, Institut für Biochemie, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany Institut für Physikalische und Theoretische Chemie, Goethe-Universität Frankfurt am Main, Max-von-Laue-Str. 7, 60438 Frankfurt am Main, Germany d Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, Germany e PRESTO, Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Saitama 332-0012, Japan b c
a r t i c l e
i n f o
Article history: Received 4 May 2016 Accepted 15 June 2016 Available online 21 June 2016 Keywords: Methanogenesis N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase Membrane protein complex SEC-MALS LILBID-MS Dimethyl maleic anhydride
a b s t r a c t Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyltetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na+ across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430 kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS. © 2016 Published by Elsevier B.V.
1. Introduction Methanogenic archaea annually produce ca. 1 billion tons of methane - an important greenhouse gas and energy resource - in anoxic habitats, such as water sediments, rice fields, hydrothermal vent peripheries as well as intestinal tracts of animals [1]. Acetate, CO2, methanol and few other one-carbon substrates are covalently bound in different oxidation states to the three one-carbon carriers methanofuran, tetrahydromethanopterin (H4MPT) and coenzyme M (HS-CoM) and reduced to methane by various electron donors [1]. The only ion-gradient forming process universally applied in the energy metabolism of methanogenic archaea is accomplished by N5-methyl-H4MPT: HS-CoM methyltransferase (Mtr). This multi-subunit membrane protein complex catalyzes the transfer of the methyl group from methyl-H4MPT to
⁎ Corresponding authors. E-mail addresses: [email protected] (S. Shima), [email protected] (U. Ermler).
http://dx.doi.org/10.1016/j.bbamem.2016.06.011 0005-2736/© 2016 Published by Elsevier B.V.
coenzyme M coupled with the vectorial translocation of Na+ across the cytoplasmic membrane [2–4]. Naþ
CH3 ‐H4 MPT þ H‐S‐CoM → CH3 ‐S‐CoM 0 þ H4 MPT ΔG ° ¼ −30 kJ=mol The Mtr complex was first isolated ca. 20 years ago from Methanothermobacter marburgensis (formerly known as Methanobacterium thermoautotrophicum strain Marburg) [5] and Methanosarcina mazei strain Gö1 [6]. The membrane fraction of M. marburgensis was extracted with 2.5% dodecyl-β- D -maltoside and the solubilized Mtr complex purified via chromatography on DEAE sepharose, Q sepharose, Superose 6 and Mono Q. The membrane-spanning complex is composed of eight subunits with molecular masses (Mr) of 25.6 kDa for MtrA, 10.7 kDa for MtrB, 27.1 kDa for MtrC, 22.8 kDa for MtrD, 31.2 kDa for MtrE, 7.3 kDa for MtrF, 9.5 kDa for MtrG and 33.5 kDa for MtrH of M. marburgensis. The apparent Mr of the whole enzyme complex was determined to be 670 kDa by size exclusion chromatography [7]. This finding led to the conclusion
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that the Mtr complex is a tetramer of heterooctamers Mtr(ABCEDFGH)4. Primary structure analysis indicates that MtrC, MtrD and MtrE are integral membrane proteins with seven, six and six membrane-spanning helices, respectively. MtrE potentially hosts a highly conserved zinc binding motif Asp-X-Glu-X22-His-X9-Glu [8] which is characteristic for all known Zn2+ binding enzymes catalyzing thiol alkylation [9,10] and an invariant aspartate in the membrane matrix which has been already reported as essential residue in several Na+ or H+ pumping protein complexes [4,11,12]. MtrA, MtrB, MtrF and MtrG only contain one transmembrane helix anchor. The soluble domain of MtrA is directed to the cytoplasm and contains an 5-hydroxy-benzimidazolyl-cobamide cofactor [5], also called factor III, which is a homologue to vitamin B12 (5, 6-dimethyl-benzimidazolyl-cobamide). Active Mtr requires cobalt of 5-hydroxy-benzimidazolyl-cobamide in the oxidation state I. MtrH is the only subunit of the Mtr complex without a membrane-spanning region and can be easily lost during cell lysis and solubilization [5]. Based on the obtained molecular and kinetic data, the reaction proceeds by a methyl transfer of methyl-H 4MPT bound to MtrH to cob(I)alamin bound to MtrA [13] thereby forming methylcob(III)alamin containing the methyl group and a histidine as axial ligands. Methyl-cob(III)alamin transfers its methyl group to coenzyme M forming methyl-coenzyme M and returns to the cob(I)alamin state. The latter reactions were shown to be dependent on Na+ [14,15] and might be therefore coupled with its vectorial translocation. However, for unravelling its complicated catalytic mechanism, larger amounts of a homogeneous protein sample and more detailed structural information are required. As a first step we developed a procedure to isolate a highly pure and homogeneous MtrA-G complex from M. marburgensis and characterized the prepared multisubunit membrane complex in terms of the absolute molecular mass and the oligomeric state. 2. Materials and methods 2.1. Materials M. marburgensis was obtained from the Deutsche Sammlung für Microorganismen (Braunschweig, Germany). The archaeon was grown as described previously [16]. Dodecyl β-D-maltoside (DDM) and dodecyl tricosaoxyethylene glycol ether (Brij-35) was purchased from Anatrace and dimethyl maleic anhydride (DMMA) from SigmaAldrich. All the chromatographic columns and column materials were acquired from GE Healthcare Lifesciences.
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final protein concentration of 200 μg/ml followed by adding solid DMMA (2 mg/ml) to the suspension. The pH of the suspension was kept at 8.0 by adding 1 M NaOH solution while continuously stirring. When the pH became constant and almost all DMMA was dissolved, the membrane was pelleted by centrifugation at 100,000 × g for 1.5 h. The membranes were then resuspended in 14 ml solubilization buffer (50 mM MOPS-NaOH pH 7.0, 10 mM MgCl2, 2 mM dithiothreitol and 2.5% w/v DDM) and incubated at 4 °C for overnight. The nonsolubilized membrane was pelleted by centrifugation at 100,000 × g for 1.5 h. The supernatant containing the solubilized MtrA-G complex was loaded to a DEAE-sepharose column (10 ml) previously equilibrated with purification buffer (50 mM MOPS-NaOH pH 7.0, 100 mM NaCl, 10 mM MgCl 2, 2 mM dithiothreitol) and 0.1% w/v DDM. The proteins were eluted with a linear 150 ml gradient of 0.1–1.0 M NaCl. The fractions containing the MtrA-G complex were selected for SDS-PAGE analysis. Size exclusion chromatography on a Superose-6 column was finally performed with the purification buffer supplemented with 0.1% w/v DDM and 10% glycerol for removing any remaining impurities and soluble aggregates and to evaluate the homogeneity of the purified complex. Detergent exchange to Brij-35 was achieved by washing and eluting MtrA-G with purification buffer containing 0.2% Brij-35 on the anion exchange DEAE-sepharose column. Purification was completed with size exclusion chromatography.
2.3. Laser induced liquid beam ion desorption mass spectrometry MtrA-G was transferred into 20 mM Tris, pH 7.0, 2 mM dithiothreitol and 0.1% w/v DDM with ultrafiltration prior to LILBID-MS analysis [17]. The spectra were recorded twice. The ion source was a commercial droplet dispenser (Microdrop), which injects microdroplets of 50 μm radius and 65 pl volume from pressure reduction apertures into high vacuum. The droplets were irradiated one-by-one by high intensity mid-IR laser pulses (wavelength ca. 3 μm) from a home-built Nd:Yag pumped LiNbO3 optical parametric oscillator. The mass analysis was performed by a home-built time of flight mass spectrometer with a Wiley-McLaren-type acceleration region and an ion reflector (Reflectron). The ions with large m/z (up to ~ 10 6) were detected by a home-built Daly-type high mass detector. Only 200 droplets were sampled for each mass spectrum. 10 μl of sample (ca. 9.0 mg/ml) was used in each analysis.
2.2. Protein production
2.4. Multi-angle light scattering
2.2.1. Protein isolation The MtrA-G complex was isolated from the membrane fraction of M. marburgensis cell as described earlier [16] with some modifications. All the steps were performed at 4 °C, if not stated otherwise. 10 g of frozen cells were thawed and resuspended in 50 ml lysis buffer (50 mM MOPS-NaOH pH 7.0, 10 mM MgCl2 and 2 mM dithiothreitol) containing 5 mg bovine pancreatic DNase I (Roche). Cells were disrupted by passing them through a microfluidizer (Microfluidics) for 4 times at 16,000 psi pressure. The cell lysates were centrifuged twice at 10,000 ×g for 30 min to remove cell debris. The membrane was pelleted from the supernatant by centrifugation at 100,000 ×g for 1.5 h, resuspended in the lysis buffer and pelleted again. After resuspending in the lysis buffer to the final volume of 5 ml, the membrane pellet was either used immediately for MtrA-G preparation or shock frozen in liquid nitrogen before storing it at −80 °C till further use.
Multi-angle light scattering coupled with size exclusion chromatography (SEC-MALS) was performed on a TSKgel G4000SWxl column (Tosoh Biosciences) at a flow rate of 0.4 ml/min on a Jasco HPLC unit (Jasco Labor und Datentechnik) connected to a light scattering detector measuring at three angles (miniDAWN TREOS, Wyatt Technology) and a refractive index detector (Optilab T-rEX, Wyatt Technology). The column was equilibrated for at least 16 h with purification buffer supplemented with 0.1% w/v DDM and 10% glycerol. The buffer was filtered through 0.1 μm pore size VVLP filters (Millipore) before applying to the column. 200 μl of protein samples (ca. 1.6 mg/ml) were separated on the column. Data analysis was accomplished using the ASTRA software package 5.3.4.13 (Wyatt Technology) and data fitting was done with a Zimm's model [18]. The refractive index increment dn/dc values of the protein and of DDM were taken as 0.185 ml/g and 0.133 ml/g respectively from the literature. The extinction coefficients of individual Mtr subunits at 280 nm were calculated using ProtParam server [19] whereas that of corrinoid prosthetic group was taken from literature [20]. The sum of the extinction coefficients of individual subunits and cobalamin (866 ml/g cm−1) was used in the data analysis.
2.2.2. Protein preparation of the MtrA-G complex after DMMA treatment The membranes from 10 g cells were resuspended at room temperature in extraction buffer (50 mM HEPES-NaOH pH 8.0, 10 mM MgCl2, 150 mM NaCl and 2 mM dithiothreitol) to adjust the
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3. Results and discussion 3.1. Preparation of a homogeneous MtrA-G complex The aerobic purification of the MtrA-H or MtrA-G complexes from the membrane fraction of M. marburgensis started from a former protocol developed for an isolation under strictly anoxic conditions. [5] To preserve the catalytic activity of the Mtr complex cobalt of 5hydroxybenzimidazolyl-cobamide has to be kept in a highly O2 sensitive oxidation state I. Despite its relative purity and integrity, the obtained Mtr complex was not sufficiently homogeneous for serious structural studies. The major problem was the loss of substantial amounts of MtrH from the membrane fraction essentially during cell lysis and membrane protein solubilization. Although the separation of MtrA-G from MtrA-H and MtrH was, in principle, possible during chromatographic purification on DEAE-sepharose [13] the amount of pure MtrA-G was very small. Extensive attempts were performed either to completely remove the non-covalently attached MtrH or to quantitatively maintain its association. However, MtrH was lost during the cell rupture independent of the used techniques although microfluidization kept the integrity of the MtrA-H complex to a higher extent. Dissociated MtrH was clearly detectable in the cytoplasmic fraction of the lysed M. marburgensis cells by SDS-PAGE and Western Blot analysis using monoclonal antibody specific to MtrH from M. marburgensis (Fig. S1). For obtaining a pure MtrA-G complex or to clearly separate MtrA-G and MtrA-H with a sufficient yield, purification was performed by exchanging the detergent DDM (CHAPS, Triton-X100, β-octylglucoside, LDAO, cholate), by variations in salt concentration (0.1–2.0 M NaCl) and pH (4.0–9.0) and after a high temperature incubation step (60 °C). Unfortunately, all these attempts failed. The breakthrough was achieved by using mild membrane perturbing agents such as low concentrations of urea or guanidine hydrochloride, alkaline pH, chaotropic salts and dicarboxylic anhydrides which were applied in the membrane-integrated state before solubilization. The most suitable agents was finally the dicarboxylic anhydride DMMA which selectively modifies at pH 8.0–9.0 free amino groups of lysines solely in water-accessible regions resulting in the disturbance of ionic interactions between the membrane-embedded and associated soluble subunits [21]. DMMA almost quantitatively detaches the water-accessible subunits of Mtr but also of other membrane proteins (facilitated further purification) and thereby does not disrupt the MtrA-G complex. An analogous result was reported for several other membrane proteins with associated soluble subunits [22–24].
Subsequent isolation steps were carried out at pH 7.0 where the modified lysine-DMMA adducts are unstable and revert back to the free amino groups. Notably, MtrH released after DMMA treatment remains intact and could be purified from the soluble fraction by ion exchange and size exclusion chromatography (Fig. S1). After solubilization of MtrA-G with DDM the enzyme complex was purified by anion exchange followed by size exclusion chromatography, instead of four steps as described in the original protocol. The MtrA-G complex was eluted in a nearly homogeneous form after passing the final size exclusion column (Fig. 1A). The yield of purified MtrA-G complex was ca. 1 mg per 10 g of wet cell mass as determined by standard Bradford assay. The Coomassie stained gel after SDS-PAGE again showed the presence of all MtrA-G subunits and the almost complete removal of MtrH (Fig. 1B). The presence of the individual Mtr subunits was ascertained by peptide mass fingerprinting analysis using for digestion either trypsin or chymotrypsin (Table S1 and S2). The success of the used method becomes obvious when comparing the MtrH content with and without treatment with DMMA (Fig. 1B and Fig. S2). Although the quality of the purification was sufficient for the subsequent molecular mass determination we continuously worked on a further improvement of the homogeneity of the protein complex for further structural studies and found out that that a detergent exchange from DDM to Brij-35 provides a more symmetric gel filtration profile (Fig. S3). 3.2. Molecular mass determination The apparent Mr of MtrA-H in the presence of 0.1% DDM was determined to ca. 670 kDa (similar to thyroglobulin) on the basis of the elution time of size exclusion chromatographic runs [5]. This value suggested a tetrameric oligomeric state because the total Mr of the individual subunits of Mtr was calculated to be 155 kDa based on a SDS-PAGE profile. Subunit MtrB with a Mr of 12.5 kDa was originally not identified as part of the Mtr complex but its consideration does not change the result [5,7]. Size-exclusion based Mr determination is fairly reliable for soluble, sphere-shaped and non-aggregating proteins but for solubilized membrane proteins rather the Mr of the protein/detergent complex than that of the protein is obtained. The amount of the bound detergent is normally unknown [25]. Methods as LILBID-MS (laser induced liquid bead ion desorption mass spectrometry) [17], SEC-MALS (size exclusion chromatography-multiple angle light scattering) [26] and sedimentation equilibrium analytical ultracentrifugation [27] provide a more reliable Mr determination as their results are less shape- and detergent
Fig. 1. Purification of the MtrA-G complex. SEC profile (A) and Coomassie stained SDS-PAGE gel (B) of the purified complex. MtrA-G in DDM was eluted at ca. 1.39 ml from a 2.4 ml Superose-6 column. Lane 1: Molecular mass marker, lane 2: Purified MtrA-G sample. Bands above 32.5 kDa in size were higher oligomers of various subcomplexes of Mtr subunits as confirmed with peptide mass-fingerprinting.
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biased. Therefore, we determined the Mr of the homogeneous MtrA-G complex in the presence of 0.1% DDM by LILBID-MS and SEC-MALS. LILBID mass spectra were recorded with two different intensities of the mid-IR laser resulting in different degrees of dissociation. In the soft detection mode at very low laser intensity (Fig. 2A) the seven prominent m/z signals carrying 1 to 7 negative charges corresponded to a Mr of ca. 430 kDa. Although LILBID-MS can resolve mass differences of ca.
Fig. 2. LILBID mass spectra of MtrA-G at low (A) and high intensity (B and C) IR laser. Both experiments were performed two times with the samples from different preparations. An Mtr species with a Mr of ca. 430 kDa was dominant in the spectrum recorded at low intensity IR laser. At high intensity IR the Mtr complex dissociated into several subcomplex species and individual subunits. The Mr of the MtrA-G (sub)complexes are taken from the left side of the peak position. The size of the shoulder of the peaks indicates a variable content of attached small molecule dominated by DDM micelles. Letters A-H in (C) are abbreviations for MtrA-H.
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100 Da, non-covalently bound smaller molecules such as cobalamin, lipids, detergents, solvents (buffer, water, metal ions) and eventually post-translational modifications substantially broaden the peak. Therefore, the Mr is read out at the external left side of the peak containing the protein fraction and firmly attached small molecules [28]. The Mr is ca. 60–80 kDa higher at the external right side of the peak essentially due to the associated detergent micelle. A second signal of the MtrA-G complex with an Mr of 504 kDa was based on three m/z peaks with a width of ca. 60–70 kDa. Under harsher conditions (high intensity IR laser pulses, Fig. 2B + C) the MtrA-G complex disintegrated into several subcomplexes and individual subunits. Broad peaks are detectable at ca. 144 kDa, 107 kDa, 82 kDa and 73 kDa. More narrow but significantly high peaks were further visible at ca. 59 kDa, 54.5 kDa and 50 kDa. The complete decomposition and the presence of all subunits could be clearly demonstrated (Fig. 2C) at harsher conditions thereby confirming the results of peptide mass fingerprinting analysis (Table S1 and S2). SEC-MALS analysis yielded a weight averaged molecular mass (Mw) for the MtrA-G complex with and without the bound detergent of 578 and 428 kDa, respectively (Fig. 3) calculated on the basis of the threedetector method using the ASTRA software. The number averaged molecular mass (Mn) of the MtrA-G complex was very close to that of Mw, being 575 kDa and 428 kDa for the MtrA-G complex with and without bound detergent, respectively. Therefore, the polydispersity index (Mw/Mn) was close to unity indicating a high monodispersity of the protein sample. This finding is in line with the narrow elution peak of SEC (Fig. 3). Both, SEC-MALS and LILBID data recorded with a soft IR-laser resulted in a dominant first species with a Mr of the MtrA-G complex of ca. 430 kDa. Considering a genome sequence-based Mr of 134.2 kDa for the MtrA-G protomer, a trimeric oligomerization of the Mtr(A-G) complex appears to be most likely under the prerequisite that all subunits are present in a 1:1 stoichiometry. The discrepancy between the theoretical and measured Mr of ca. 27 kDa (430 kDa − 3 × 134.2 kDa) are most likely due to firmly bound small molecules composed of cobalamin, structural lipids, solvents and DDM. The value of ca. 8–10 kDa of small structural molecules per 100 kDa membrane protein is in line with the findings in AcrB [28] and virtually in complex I [29] but differ from those of ATPases [30,31].
Fig. 3. Molecular mass analysis of MtrA-G complex with SEC-MALS. MtrA-G was subjected to SEC-MALS analysis for the determination of its absolute Mr without detergent. The experiment was performed two times. The left axis represents Mw. Mn was calculated with the ASTRA software. The Mw and Mn of the protein fraction of the proteindetergent complex were calculated to 428 kDa giving the polydispersity index close to unity. The Mr of the protein-detergent complex, the protein and the detergent are marked with a black, red and blue line.
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A second species present in small amounts has a Mr of 504 kDa and 575 kDa based on LILBID-MS and SEC-MALS experiments, respectively. We primarily interpreted the excess mass as detergent micelles bound differently strong to the enzyme although the presence of minor amounts of MtrH has also been considered. When integrating the small structural molecules the Mr ratio protein/detergent of MtrA-G obtained by LILBID-MS is 5.8 (430 kDa/504–430 kDa) and by SEC-MALS 2.9 (430 kDa/580–430 kDa). Notably, the apparent Mr of 670 kDa reported for the MtrA-H-detergent complex [7] is also compatible with a trimeric oligomerization state. The Mr ratio protein/detergent is 3.0 (534 kDa/670–534 kDa) with the Mr of the MtrA-H trimer (3 ∗ 178 kDa) being composed of the mass derived from the gene sequence (168 kDa) and from small structural molecules (estimated to ca. 10 kDa). The application of harsher IR laser conditions resulted in the formation of subcomplexes (Fig. 2B). The peak at 144 kDa can be assigned to MtrA-G indicating that the protomer assembles to a compact, fairly interconnected unit, which less strongly interacts with the other protomers. Moreover, the discrepancy between measured and theoretical Mr (134.3 kDa) of ca. 10 kDa corresponds to the value for the entire (MtrA-G)3 complex per protomer. This extra mass is attributed to firmly attached small structural molecules and not as one of the small subunits resulting in a stoichiometry of the subunits MtrA-G of 1:1. The broad peak at ca. 107 kDa might correspond to either the MtrBCDEFG (108.7 kDa) protomer without the partly soluble MtrA or less likely the MtrABDEFG (107.1 kDa) subcomplex missing the membrane subunit MtrC. The MtrBCDEFG assignment is in line with the peak at ca. 82 kDa assigned as the MtrCDE (81 kDa) membrane core complex. Their significant peak width still indicates a considerable amount of attached small molecules. More narrow peaks at ca. 73, 59, 54.5 and 50 kDa can be tentatively assigned to MtrAEFG (73.7 kDa), MtrCDG (59.4), MtrBCFG (54.7 kDa) and MtrCD (49.9 kDa), respectively (Fig. 2B). Although the number and the composition of the detected subcomplexes do not convey a sufficiently complete picture about the subunit organization within the MtrA-G protomer, we tentatively constructed a preliminary schematic model (Fig. S4). Other techniques such as crosslinking and subsequent protease digestion peptide mass fingerprinting experiments as well as detailed structural methods are necessary for substantiation. The highly pure and homogeneous MtrAG complex provides a serious platform for these studies. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements We thank Rudolf K. Thauer and Hartmut Michel for continuous support. V.U. is grateful to the International Max Planck Research School for funding. We are grateful to Volker Müller for providing us with antibodies against ATP synthases from various methanogens. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamem.2016.06.011. References [1] R.K. Thauer, Biochemistry of methanogenesis: a tribute to Marjory Stephenson, Microbiology 144 (1998) 2377–2406. [2] B. Becher, V. Müller, G. Gottschalk, N5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase of Methanosarcina strain Gö1 is an Na+-translocating membrane protein, J. Bacteriol. 174 (1992) 7656–7660.
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