NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1) Ulrich Matthey 1 , Daniel Braun 2 and Peter Dimroth 1 1 Institut fu ¨ r Mikrobiologie, and 2 Institut fu ¨ r Molekularbiologie und Biophysik, Eidgeno ¨ ssische Technische Hochschule, Zu ¨ rich, Switzerland The subunit c from the A TP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/ water (4 : 4 : 1). In this s olvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G 29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the a helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the p rominent interruption of the a helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the a helix after the sodium ion-binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent a lso d eviated from that o f Escherichia coli in the same environment, the most important difference being that sub- unit c of P. modestum did not adopt a stable hairpin struc- ture like subunit c of E. coli. Keywords: ATP synthase; stable isotope labeling, NMR spectroscopy; Propionigenium modestum; subunit c. F 1 F 0 ATP synthases catalyse the formation of ATP from ADP and inorganic phosphate that is driven by an electrochemical gradient of protons or in some cases Na + ions. Similar e nzymes are found in chloroplast, mitochon- dria and bacteria. They consist of a cytoplasmic F 1 part with the subunit composition a 3 b 3 cde and a membrane intrinsic F 0 moiety, which in bacteria has the subunit composition ab 2 c x . The mechanism for ATP synthesis, proposed to involve binding changes of the three catalytic binding sites on the b sunbunits [1], was in remarkable agreement with the atomic resolution X-ray structure of F 1 [2]. Based on these data rotation of subunit c within the cylinder made of alternating a and b subunits was suggested and confirmed [3–5]. More recent structural data have shown that the c and e subunits forming the central stalk are permanently fixed to the ring of c subunits [6], and consequently, all three subunits were demonstrated to rotate as a unit [7–9]. A high-resolution structure of the ion-translocating F 0 part remains to b e determined. Electron and atomic f orce microscopy of F 0 indicated that subunits a and b a re attached to the periphery of an oligomeric ring of c subunits [10,11]. The subunits a and c a re directly involved in ion translocation [12–15], whereas subunit b is presumed to form a peripheral stalk, which connects the F 0 part to F 1 via associationwiththed subunit [16–18]. Based on structu ral data, the number of c subunits forming the ring is c 10 for the yeast ATP synthase [6], c 14 for t he chloroplast enzyme [19], and c 11 for the Ilyobacter tar taricus ATP s ynthase [20]. According to cross-linking studies, c 10 appears to be the preferred stoichiometry for the ATP synthase of Escherichia coli [21]. The NMR structure of the monomeric E. coli subunit c was determined in chloroform/methanol/water (4 : 4 : 1), in which the protein folds like a hairpin [22,23]. Two extended a helices are connected by a hydrophilic loop, and the proton-binding residue D61 is located in the centre of the C-terminal helix. The monomeric P. modestum sub- unit c was studied by NMR in SDS micelles [ 24]. In this biphasic system the protein consists of four a helical segments, t hat are connected by short linker peptides with nonregular secondary structures. The Na + -binding residues Q32, E65 and S66 [12] are lo cated in the I–II and III–IV helix connections. No lo ng-range NOEs could be identified that would indicate the presence of a three-dimensional fold with close packing of the helices. In order to enable a direct comparison of the P. mode- stum subunit c with its E. coli homologue, we now prepared an NMR sample in chloroform/methanol/water (4 : 4 : 1). The key questions to be investigated were how the secondary structure in the organic solvent mixture compares with that in dod ecylsulfate mic elles, and w hether the P. modestum subunit c forms similar interhelix contacts as the E. coli protein in the same solvent. EXPERIMENTAL PROCEDURES Overproduction and purification of subunit c Unifo rml y 13 C, 15 N-labelled subunit c was overproduced in E. coli PEF42(DE3)pT7c on Martek 9-CN medium as described previously [24]. Subunit c was purified by chloroform/methanol extraction and anion-exch ange Correspondence to U. Matthey, Institut fu ¨ r Mikrobiologie, Eidgeno ¨ ssische Technische Hochschule Zu ¨ rich, ETH-Zentrum, CH 8092 Zu ¨ rich, Switzerland. Fax: + 41 1632 13 78, Tel.: + 41 1632 55 23, E-mail: matthey@micro.biol.ethz.ch Enzymes:H + -transporting ATP synthase (EC 3.6.1.34); Na + -transporting ATP synthase (EC 3.6.1.37). Note: web page available at h ttp://www.micro.biol.ethz.ch (Received 3 December 2001, r evised 18 February 2001, accepted 20 February 2001) Eur. J. Biochem. 269, 1942–1946 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02851.x chromatography [25]. Aliquots of 4 mL were applied to a Sephadex LH20 column (160 mm · 20 mm) and eluted with chloroform/methanol (2 : 1). Fractions of 3 mL were collected and the protein was monitored by A 280 . NMR sample preparation Subunit c was transferred into C 2 HCl 3 /C 2 H 3 OH/H 2 O (4 : 4 : 1), applying the same conditions as for the E. co li protein (M. Girvin, Biochemistry Department, Albert Einstein College of Medicine, New York, USA, personal communication). Ten milliliters of C 2 HCl 3 were added to 11 mL of subunit c [1 mgÆmL )1 in chloroform/methanol (2 : 1)] and the mixture was concentrated to less than 0.1 m L by a gentle stream of Argon with periodical swirling of the s olution to k eep the s olvent composition uniform. After addition of 0.5 mL C 2 HCl 3 /C 2 H 3 OH (2 : 1), the sample was incubated for 15 min at room temperature, and then 0.4 mL C 2 HCl 3 was added. The sample was concen- trated to 0.05–0.1 mL and the same c ycle of solvent addition and subsequent volume reduction was repeated. The sample was then brought to complete dryness with a stream of Argon. Whenever the sample became cloudy during the preparation, C 2 HCl 3 wasaddedtoredissolvethe material. The dried sample was covered with 0.6 mL of C 2 HCl 3 /C 2 H 3 OH/H 2 O containing 25 m M D 11 -Tris/HCl at pH 7.5, and incubated for 1 h at 30 °C with periodical, gentle swirling. After adjusting the pH to 7.5, the sample was centrifuged and transferred to an NMR tube, w hich was flame-sealed. The final concentration of the protein was 2m M . NMR spectroscopy Spectra were recor ded at 27 °C o n B ruker D RX500, DRX600, DRX750 and DRX800 spectrometers. For the resonance assignments and the collection of conformational constraints, the following experiments were recorded: 3D CBCA(CO)NH [26], 3D HNCACB [27], 3D 15 N-resolved [ 1 H, 1 H]-TOCSY (mixing time s m ¼ 60 ms) [28], 3D 13 C-resolved HCCH-TOCSY (s m ¼ 14 ms) [29], 3D 15 N-resolved [ 1 H, 1 H]-NOESY (s m ¼ 60 ms) [ 30], 3D 13 C- resolved [ 1 H, 1 H]-NOESY (s m ¼ 60 ms) [31], and 3D 13 C- resolved [ 1 H, 1 H]-NOESY (s m ¼ 150 ms). Spectra were processed and analysed with the programs PROSA [32] and XEASY [33]. Chemical shifts were calibrated with sodium 3-(trimethylsilyl)propane-1-sulfonate. RESULTS Stability of P. modestum subunit c in chloroform/methanol/water (4 : 4 : 1) Samples of 2 m M unlabelled subunit c of Propionigenium modestum in C 2 HCl 3 /C 2 H 3 OH/H 2 O (4:4:1), 25m M D11-Tris/HCl were prepared to test the stability of the protein. Two-dimensional homonuclear NMR spectra were recorded to monitor structural changes over 4 weeks. During this period, no spectral differences were observed with samples at pH 5.8, pH 7.0 and pH 7.5 that were kept at 20 °C. These observations indicated that P. modestum subunit c was stable in the solvent mixture at the pH values indicated. Resonance assignment Sequence-specific backbone assignments f or subunit c were obtained from 3D HNCA, 3D CBCA(CO)NH and HNCACB experiments using a 2-m M 15 N/ 13 C-labelled sample in chloroform/methanol/water (4 : 4 : 1). With the exception of the N-terminal dipeptide se gment H-Met-Asp- all amide protons and nitrogen resonances could be assigned. Proton resonances of aliphatic side chains were achieved by 3D 15 N-resolved [ 1 H, 1 H]-TOCSY and 3D 15 N- resolved [ 1 H, 1 H]-NOESY. Furthermore, homonuclear 2D [ 1 H, 1 H]-TOCSY and 2D [ 1 H, 1 H]-NOESY spectra were used to assign the aromatic spin systems. While all aromatic 1 H s ide chain resonances of Y34, Y70 and Y80 could be determined, only one aromatic side chain resonance was found for F84 due to signal overlap. The 13 C chemical shifts were obtained by 3D 13 C-resolved HCCH-COSY, 3D 13 C-resolved HCCH-TOCSY and 3D 13 C-resolved [ 1 H, 1 H]-NOESY experiments. However, chemical shift dispersion of the methyl groups was limited, complete assignment of all CH n groups was received except those of the N-terminal methionine. Conformational constraints Overall 3035 NOESY cross-peaks were assigned. We found 515 intraresidual, 283 sequential and 331 medium-range NOEs. Observed d ad NOEs showed that the peptide bonds G27–P28, Q46–P47 a nd N82 –P83 are all in trans-confor- mation. No long-range NOEs could be identified in the spectra recorded with a mixing time of 60 ms that would indicate the occurrence of close interhelix contacts. There- fore, additional 2D [ 1 H, 1 H]-NOESY and 3D 13 C-resolved [ 1 H, 1 H]-NOESY spectra with 150 ms mixing time were recorded on a Bruker DRX800 spectrometer and analysed. Possible long-range NOEs were collected using the chemical shift comparison function of CANDID. Evaluation of these signals did not indicate the presence of any long-range NOEs. In particular, cross-peaks of aromatic protons could be completely assigned as short-range and medium-range NOEs (Fig. 1 ). The secondary structure characteristic connectivities were collected from NOESY spectra recorded with 60 ms. Surprisingly, the proposed hydrophilic loop between Q46 and D52 showed significant a helix con nectivities (Fig. 2). In contrast to previous NMR studies of P. modestum subunit c in SDS micelles [24], subunit c in chloroform/ methanol/water (4 : 4 : 1) exhibited no ahelix interruption at t he C-terminal part (T67, G68) of the protein. Similar to the SDS sample, subunit c in chloroform/methanol/water (4 : 4 : 1) contains a nonhelical linker peptide near P28, which is shorter t han in the SDS structure. Overall, typical a helix connectivities were f ound from L5 to I26, from G29 to Q46 and from P47 to N82. A s indicated by t he d aN (i,i + 2) connectivities the two helices possibly end with 3 10 helix turns comprising residues I23 to I26 and N82 to L87, respectively. Chemical shift deviations The deviations from the random coil chemical shifts were calculated as the difference b etween the measured chemical shifts and the corresponding random coil values in aqueous Ó FEBS 2002 NMR studies of P. modestum subunit c (Eur. J. Biochem. 269) 1943 solution [34]. Continuous 13 C a downfield chemical shift deviations were found from V4 to G23, V30 to A44 and I53 to Y80 (Fig. 2). Significantly smaller deviations were observed for the peptide segments G25 to G29, R45 to D52 and A81 to G89. The shape of the chemical shift deviations did not change when calculated with corre- sponding random coil values in chloroform/methanol (1 : 1), whic h w ere referenced with tetramethylsilane (H. K essler, Institute fu ¨ rOrganischeChemieandBio- chemie, Tu M u ¨ nchen, Garching, Germany, personal communication). However, the chemical shift deviations were about 1.9 p.p.m. higher, which can most probably be attributed to the usage of different refer ence stan- dards. Secondary structure and global fold As indicated by helix-characteristic NOE connectivities [35], subunit c in chloroform/methanol/water (4 : 4 : 1) consists of two helices. Helix I comprises residues L5 to I 26, and helix II comprises the segment G 29 to N82 with a short interruption between Q46 and P47. In particular no signal intensities were obtained for the a helix characteristic connectivities d aN (23,26), d ab (23,27), d aN (24,27), d ad (24,28), d aN (25,29), d ab (25,28), d ad (25,28) and d aN (26,30). The interruption between helix I and helix II is further confirmed by the small 13 C a downfield chemical shift deviation of the segment A24 to G29. The proposed hydrophilic loop Q46 to D52, is a helical according to the medium-range NOEs. However, the 13 C a chemical shift deviation of this peptide segment is signifi- cantly smaller than that of the other helical segments. Therefore, it is likely that the a helix conformation of this segment is poorly populated. DISCUSSION Structural studies of membrane proteins by NMR in solution require the usage of either organic solvents or detergents. T he s tructure o f the E. coli s ubunit c was studied in organic s olvent by NMR [23]. Addition of water to E. coli subunit c in chloroform:methanol (1 : 1) was found to stabilize interhelix contacts in a concentration depending manner (M. Girvin, Biochemistry Department, Albert Einstein College of Medicine, New York, USA, personal communication). In chloroform/methanol/water (4 : 4 : 1) the protein consisted of two elongated helices, which w ere connected by a h ydrophilic loop. Two different structures at pH 5 and pH 8 were found and a conforma- tional change of subunit c during ion translocation was proposed [22]. The secondary structure of subunit c of P. modestum in SDS micelles [24] deviates significantly from that of E. coli in chloroform/methanol/water (4 : 4 : 1): P. modestum sub- unit c folds into four helices, that are connected by small linker peptides with nonregular secondary structure. The Na + -binding ligands (Q32, E65, S66) [12] are located in the peptides connecting helices I and II, and III and IV, respectively. As described a bove, the two NMR s tructures were determined in different solvent system s and for t he c subunits from two different b acteria. It w as therefore intriguing to investigate whether the structure of the c subunit is dependent on the solvent conditions. For this purpose, we determined the structure of subunit c from P. modestum in chloroform/methanol/water (4 : 4 : 1). Under these conditions, the protein f olds significantly dif- ferent from subunit c in dodecylsulfate micelles (Fig. 3B,B¢) and it also folds significantly different than subunit c of E. coli in the organic solvent mixtures ( Fig. 3A). The secondary structure of P. modestum subunit c in the organic solvent is mainly helical with only a short linker peptide consisting of residues G27 and P 28, but without interrup- tion of the helical folding in t he hydrophilic loop region as indicated b y h elix-characteristic NOEs. Subunit c of P. modestum therefore does not fold into a s table helical hairpin in chloroform/methanol/water (4 : 4 : 1). The hair- pin structure of subunit c is indicated, however, by a wealth of experiments [12,36–40] and was unequivocally demon- strated by the F 1 c crystal structure from yeast mitochondria [6]. Moreover, the inconsistency between chemical shift deviations and NOE connectivities at the peptide seg- ment R45 to D52 implies structural polymorphism. The P. modestum subunit c showed structural variety in differ- ent solvent systems. It is likely that small changes i n the organic solvent composition can cause structural polymor- phism. Further, homologue proteins (P. modestum and E. coli subunit c) can form different structures in the same solvent, depending on their individ ual sequences. Fig. 1. Part of a contour plot of a two-dimensional [ 1 H, 1 H]-NOESY spectrum showing the methyl-aromatic proton cross-peaks o f subunit c in chloroform/methanol/water (4 : 4 : 1). The spectrum was reco rded with 150 ms mixing times on a Bruker DRX800 spectrometer. Assignments of the signals are indicated. 1944 U. Matthey et al. (Eur. J. Biochem. 269) Ó FEBS 2002 We hypothesize that monomeric subunit c may be considerably prone to structural polymorphism. Regardless of whether the protein is dissolved in organic solven ts or embedded into detergent micelles, these environments are not a good mimic of the natural situation where the protein forms strong protein/protein contacts to assemble into rings of 10, 11 or 14 c subunits. Th ese rings can be extremely s table, and resist boiling in SDS for 5 min in the case of the undecameric c ring of P. modestum [41]. This stability of the ring makes structural flexibility rather unlikely, indicating that the structural polymorphism of monomeric subunit c is due to the interaction of the monomer with the artificial environment lacking the stabilizing protein/protein contacts within the ring. The undecameric c ring of the ATP synthase from Ilyobacter tartaricus, a close relative to P. modestum, has recently been crystallized in two dimensions and subjected to structure determination by cryo-transmission electron microscopy [20]. All c subunits of the ring show th e hairpin-like folding and the structure of all monomeric units appears to be the same. Hence, the structural flexibility observed for the subunit c monomer is apparently lost upon assembly into the ring, probably b ecause r ings of the stability observed require defined structures of the monomeric units in order to gen erate strong protein/protein interactions. ACKNOWLEDGEMENTS We thank Georg Kaim and Mark E. Girvin for critical reading of the manuscript. We are grateful to Torsten H errmann for his support i n CANDID, Reto H orst for introduction into the DRX800 spectrometer and Mark Girvin for providing the detailed protocol of the NMR sample preparation. We express a special thank to Kurt W u ¨ thrich for his support and the provision of NMR equipment. REFERENCES 1. Boyer, P.D. (1993) The binding change mechanism for ATP synthase – some probabilities and possibilities. Biochim. Biophys. Acta. 1140, 215–250. 2. Abrahams, J.P., Leslie, A.G.W., Lutter, R. & Walker, J.E. (1994) Structure at 2 .8 A ˚ resolution of F 1 -ATPasefrombovineheart mitochondria. Nature 370, 621–628. 3. Noij, H., Yasuda, R., Yoshida, M. & Kinosita, K. (1997) Direct ob servatio n o f t he rotation of F 1 -ATPase. Na ture 38 6, 299– 302. 4.Duncan,T.M.,Bulygin,V.V.,Zhou,Y.,Hutcheon,M.L.& Cross, R.L. (1995) Rotation of subunits during catalysis by Escherichia coli F 1 -ATPase. Proc.NatlAcad.Sci.USA92, 10964– 10968. 5. Sabbert, D., Engelbrecht, S. & Junge, W. (1996) Intersubunit rotation in active F-ATPase. Nature 381, 623–625. Fig. 3. Alignment of c subunits from E. coli and P. modestum and location of the a helical secondary structures in different subunit c preparations. (a) Amino-acid sequence of E. coli subunit c and location of a helices in the st ructure in ch loroform/meth anol/water (4 : 4 : 1) [23], amino-acid sequence of P. modestum subunit c with the helix locations in (b) SDS micelles [24], and (b¢) in chloroform/methanol/water (4 : 4 : 1 ). Identical amino ac ids are denoted by white l etters on dark gre y background and conservative substitutions are indicated by light grey background. The conserved carboxylate residues involved in ion b inding (E. coli D61, P. modestum E65) are indicated with an asterisk. Fig. 2. Amino-acid sequence of P. modestum subunit c a nd survey o f s elected N MR data. Sequential NOE connectivities are displayed b y c ontinuous bold line s extending over the residues that showed these connectivities. Medium-range NOEs are represented by lines connec ting the two in teracting residues. I n the row Dd( 13 C a ) t he difference be tween the ob served shifts and the co rrespo nding random coil values [34] is sh own. 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