Supplementary Information

Transkript

1 Supplementary Information Asp432 M1 site Asp432 Asp432 Asp432 M1 site Supplementary Figure 1 The electron density maps of the M1 site of MgtE crystal structure. Stereo view of the M1 site with the 2mFo DFc electron density map contoured at 1.2 σ. Takeda et al. Supplementary Figure 1

2 M2 M2 M3 T.therm H.salin M.mazei P.abyss S.aureu E.coli T.marit SLC41A1D1 SLC41A1D2 SLC41A2D1 SLC41A2D2 SLC41A3D1 SLC41A3D LGAVD V PDLVYSEAGPVALWLARVRWLV I L I LTGMVTSS IL QGFESV...LEA V TA LAFY...MSVRRVAATAYREAAPALA A S V LGGLFAGA VL GGMRAQ...LEA V DG LLV V MGTVG L MRTGLKDANVRLLYAKRVPWLLTL V FINIFSGA AI ASFEDT...IQA V VA LVI F VILGI L RGYSKLNREDLKF L RELLTIIGGL A LLSSITGS LL ESYSKL...IDS V AIFS V M EYSRL V SDIDSTNDSIIKT A LKRLPWLI I LTFLGMITAT IL GRFEKT...LEN V AL LAA F RRLGG L SAGEDVFAPVTKA V KTRWAWLA V N L CTAFIASR VI DGFEHT...ISQ L VA LASL MASMS A TYTSYFHTPAWKL I LKRSPWLV V L L LLESVNGN II SSYEKF...LAS I PI IAA F VSTDRGPAPPSPLKETSFS I GLQVLFPF L L A GFGTVAAG MV LDIVQHWEVFQK V TE V F I L VALLP V WVVLARRSPATRE V LYSGWEPV I I A MAISSVGG LI LDKTVS...DPNFAG MAV F DEDAI V EVTPKLPKESSGI M ALQILVPF L L A GFGTVSAG MV LDIVQHWEVFRK V TE V F I L LALTP I WIIIAAKHPATRT V LHSGWEPV I T A MVISSIGG LI LDTTVS...DPN L VG IVV Y QSVTPKPLETEPSRETAWS I GLQVTVPF M F A GLGLSWAG ML LDYFQHWPVFVE V KD LLTL AALTP V WVLIAKQSPPIVK I LKFGWFPI I L A MVISSFGG LI LSKTVS...KQQYKG MAI F L T.therm H.salin M.mazei P.abyss S.aureu E.coli T.marit SLC41A1D1 SLC41A1D2 SLC41A2D1 SLC41A2D2 SLC41A3D1 SLC41A3D2 V P VL L G TG GNTGNQSATL I IRA L ATRD L D...LKDWRRVFLKEMG V GLL V P AL LATR GNVYASFGAR L ATG L HQGI I EP...HVAAGDRRLRAAVG A AMG L P LL IDSG GNAGSQSATL V VRS L ATGD V K...NKDWFYLIGREML V AAT Y P MV LDTT GNLGSIIGAKTSTK L HLEG I EK...IINLDILKEITVYSL L ALP I P II S G MS GNSGTQSLAVSVRN I TTGE I N...EQSKFRIALREAGSGVL M P IV A G IG GNTGNQTITM I VRA L ALQN I Q...PGNLTFLILREMG V AVI I P T M I G SA GNTGAQISAL M IRGFTLNE I S...LKDWWKVLLRESL I GST V P AL L G LK GNLEMTLASR L STA A NIGH M D...TPKELWRMITGNMA L IQV T P VI N G VG GNLVAVQASR I STF L HMNG M PGENSEQAPRRCPSPCTTFFSPDVNSRS A RVL V P AL L G LK GNLEMTLASR L STA V NIGK M D...SPIEKWNLIIGNLA L KQV T P VI N G IG GNLVAIQASR I STY L HLHS I PGELPD.EPKGCYYPFRTFFGPGVNNKS A QVL V P P L V G LK GNLEMTLASR L STA A NTGQ I D...DPQEQHRVISSNLA L IQV T P VI C G VG GNLVAIQTSR I STY L HMWS A PGVLPLQMKKFWPNPCSTFCTSEINSMS A RVL TM2 TM3 M T.therm H.salin M.mazei P.abyss S.aureu E.coli T.marit SLC41A1D1 SLC41A1D2 SLC41A2D1 SLC41A2D2 SLC41A3D1 SLC41A3D2 LG L T L S.. LL L V GKVYWDG...HPLLLP V VGVS LV LI V FFANL V GAL L PF LLRR... NG LLA SGF AA A V AFGVLTALG..RPVAGLAT L VGVA LV AG L LSGVA L TGA V VA VVFAGYR IG I T M G.. AA A A GLGIYRG...GLELGV V VAVT MI IV I FAGCL I GTT L PF LLTA... LG IV GNVIG M E L TKLLLHR...KAEIIPHFILLYPVF V FSVLWFAYF L AI VADR... SG VV CSTI L FT I IVAIYH...QPLLAL I VAGS L TCA M TVGTF V GSM I PL LMNK... NG LV WGGI M GG I TWWLYDD...MALGG V MTLA MM LN L LMAAL M GVI I PMT M VK... LG LIL AG. VL Y L RAFLISS...DPTLNF A VATA LL VL I LYANI M GAL L PF IARI... QAT VV GFL A SI A AVVFGWIPDGHFSIPHAFL L CASS VA TAFIASLV L GMI M IG VIIGSRK FL LVV PGH LV F L YTISCMQGGHTTLTLIFIIFYMTA AL LQ V LILLY I ADW M VHW M WG... QAT VV GFL AA V A AIILGWIPEGKYYLDHSIL L CSSS VA TAFIASLLQGII M VG VIVGSKK LL LVI PGH LI F L YTIHLMKSGHTSLTIIFIV V YLFG AV LQ V FTLLW I ADW M VHHFWR... QAT VV GLL AA V A ALLLGVVSREEVDVAKVEL L CASS VL TAFLAAFA L GVL M VC IVIGARK LL LVV PGH LI FFYIIYLVEGQSVINSQTFVV L YLLAG L IQ V TILLY L AEV M VR L TWH... TM4 M T.therm H.salin M.mazei P.abyss S.aureu E.coli T.marit SLC41A1D1 SLC41A1D2 SLC41A2D1 SLC41A2D2 SLC41A3D1 SLC41A3D2 LG V D P ALVSN P L V AT L S D V TGLL I YLS V ARLLLEAV... RGYN P DTLVG P L V TTTG D L FGLAFLVL A VRLIVGGV... LK M D P ATASA P L I TS I A D I VGVV I YFR I ATWYYGIA... AK L D P DNVTV P T I TT L S D V FSTLFIVG I AKIVVG... LN I D P AVASG P F I TT I N D I ISML I YFG L ATSFMAYLI... LGRD P AVGSSVM I TA I T D TGGFF I FLG L ATLFLM... FK I D P AFMAG P L L TT I V D V TGIM I YFY V VHSFLS... IG I N P DNVAT P I A AS L G D L ITLA L LSG I SWGLYLELNHWRYIYPL... RG L D P DNFSI P Y L TA L G D L LGTG L LALSFHVLW...LIGDRDTDVGD... TG I N P DNVAT P I A ASFG D L ITLA I LAW I SQGLYSCLETYYYISPL... KGKD P DSFSI P Y L TA L G D L LGTA L LALSFHFLW...LIGDRDGDVGD... LG V N P DNIAT P I A AS L G D L ITLS I LAL V SSFFYRHKDS.RYLTP... QA L D P DNHCI P Y L TG L G D L LGSSSVGHTAAVPRRCTASPGWGLIQPFICTQHLIVSLLSF Takeda et al. Supplementary Figure 2

3 Supplementary Figure 2 Amino acid sequence alignment with secondary structures of MgtE. Amino acid sequence alignment of MgtE with its homologues, constructed by CLUSTALW 2 and ESPript2. 3. Abbreviations of organisms or proteins are as follows: T. therm., Thermus thermophilus; H. salin, Halobacterium salinarum; M. mazei, Methanosarcina mazei; P. abyssi, Pyrococcus abyssi; S. aureu, Staphylococcus aureus; E. coli, Escherichia coli; T. marit, Thermotoga maritima. The SLC41 proteins contain two tandemly-repeated domains (N-terminal D1 and C-terminal D2) homologous to the TM domain of the bacterial MgtE proteins. The secondary structure of TtMgtE is shown below its sequence. Strictly conserved and similar residues are represented within a red box and by a red letter, respectively. The conserved amino acid residues of the M1 site involved in ion recognition are indicated by red circles. Amino acid residues comprising the M1, M2 and M3 sites are labeled above the sequence and mutated amino acid residues are indicated by circles.

4 a b M2 site c M2 site M2 site Mn2+ Glu37 His383 Glu37 Glu37 His383 e Symmetry molecule f M3 site Symmetry molecule M3 site Glu275 Glu275 Glu311 Glu311 Glu311 His383 d M3 site Symmetry molecule Glu275 Mn2+ Glu311 Glu311 Ca2+ Supplementary Figure 3 Structure of the M2 and M3 sites (a-c) Close-up views of the M2 sites in the Mg2+- (a), Mn2+- (b) and Ca2+- (c) bound structures. The 2mFo DFc (1. ) and anomalous difference Fourier (4. ) maps are colored green and magenta, respectively. (d-f) Close-up views of the M3 sites in the Mg2+- (d), Mn2+- (e) and Ca2+- (f) bound structures. The 2mFo DFc (2. ) and anomalous difference Fourier (4. ) maps are colored green and magenta, respectively. Takeda et al. Supplementary Figure 3

5 a Fluorescence intensity (au) Mn 2+ Mg 2+ Ca 2+ b 1 / F (au ) Benesi-Hildebrand plot Ca 2+ Mg 2+ Mn Divalent cation (μm) / Divalent cation (μm -1 ) Supplementary Figure 4 Response of KMG-2 to Mg 2+. (a) Relationship between the fluorescence intensity of KMG-2 and the divalent cation concentration. The fluorescence of KMG-2 was excited at 44 nm, and emissions were monitored at 5-53 nm. (b) Benesi-Hildebrand plots for the complexation of KMG-2 with Mg 2+, Ca 2+ and Mn 2+. All data points are mean ± SEM (n=3). Takeda et al. Supplementary Figure 4

6 25 Mg 2+ uptake (μmol mg -1 protein min -1 ) Liposome WT M2A MgCl 2 (mm) Supplementary Figure 5 Mg 2+ uptake assay. Mg 2+ uptake activities of wild-type MgtE and the M2A mutant. All data points are mean ± SEM (n=3). Takeda et al. Supplementary Figure 5

7 b Fluorescence intensity (au) Liposome 4 + Nystatin-ergosterol + WT 3 + M2A 25 mm NaCl Fluorescence intensity (au) a Liposome 14 + Nystatin-ergosterol + WT 12 + M2A 1 mm KCl Time (s) Time (s) Supplementary Figure 6 Na and K uptake assays. (a, b) Na+ (a) and K+ (b) uptake activities of wild-type MgtE and the M2A mutant were measured in the absence of Mg2+. The nystatin and ergosterol complex is a non-selective monovalent cation ionophore4 used as a positive control. This experiment was repeated three times. Takeda et al. Supplementary Figure 6

8 Fluorescence intensity (au) WT (+IPTG) mm Mg 2+ WT ( IPTG) 4 2 WT (+IPTG) + 1mM Mn Time (s) Supplementary Figure 7 Time-dependent Mg 2+ accumulation. Changes in the fluorescence intensity derived from Mag-Fluo4-AM-loaded giant spheroplasts. MgCl 2 was added at the indicated time. Takeda et al. Supplementary Figure 7

9 a TM3 Glu378 Glu3 M2 site TM4 Glu378 M2 site Glu378 TM2 Glu3 M3 site Glu378 M3 site Glu3 M2 site 9 Glu3 TM2 M2 site Glu378 TM4 TM3 c A 3A M 2M M T 2A [Mg2+]out = 25 mm MnCl2 (mm) N o M n pr ot ei 3 M A 2M 3A 2A M M n T W pr ot ei WT M2A M3A M2M3A 1.2 N o 1.4 Normalized caiton uptake 1 d 4 Mn2+ uptake (μmol mg-1 protein min-1) Mg2+ uptake (μmol mg-1 protein min-1) 12 W b Supplementary Figure 8 Predicted structure and liposome-based assay of MgtE from Methanosarcina mazei (MmMgtE). (a) Predicted structure of MmMgtE, constructed by the Phyre server5. The PDB template used for modeling was 2ZY9. Hydrophilic amino acid residues are depicted by ball and stick representations. (b,c) Divalent cation uptake by wild-type MmMgtE and its mutants in the presence of 1 mm Mg2+ (b) and Mn2+ (c). (d) Competition assay with Mn2+ of wild-type MmMgtE and its mutants in the presence of 25 mm MgCl2 and the indicated concentration of Mn2+. All data points are mean ± SEM (n=3). Takeda et al. Supplementary Figure 8 1

10 Supplementary Information References 1. Hattori, M. et al. Mg 2+ -dependent gating of bacterial MgtE channel underlies Mg 2+ homeostasis. EMBO J. 28, (29). 2. Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, (1994). 3. Gouet, P., Courcelle, E., Stuart, D. I. & Métoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 35 8 (1999). 4. Kleinberg, M. E. & Finkelstein, A. Single-Length and Double-Length Channels Formed by Nystatin in Lipid Bilayer Membranes. 269, (1984). 5. Kelley, L. a & Sternberg, M. J. E. Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, (29).