Identification of critical residues of subunit H in its interaction with subunit E of the A-ATP synthase from Methanocaldococcus jannaschii Shovanlal Gayen, Asha M. Balakrishna, Goran Biukovic ´ , Wu Yulei, Cornelia Hunke and Gerhard Gru ¨ ber School of Biological Sciences, Nanyang Technological University, Singapore Energy is the ability to do work or bring about a change. Living things are constantly changing, and therefore need to acquire energy. The molecule ATP is the common energy currency of cells; when cells require energy, they ‘spend’ ATP. In the case of the archaea, the A 1 A 0 ATP synthases (A-ATP synthase) catalyse this process of ATP synthesis [1]. This class of enzyme is composed of ten subunits with the stoi- chiometry A 3 :B 3 :C:D:E:F:G:H 2 :a:c x . Like the related bacterial F 1 F 0 ATP synthase (F-ATP synthase) (a 3 :b 3 :c:d:e:a:b 2 :c x ) and the eukaryotic V 1 V 0 ATPase (V-ATPase) (A 3 :B 3 :C:D:E:F:G 2 :H:a:d:c x :c¢ x :c¢¢ x ), it pos- sesses a water-soluble A 1 domain, containing the cata- lytic sites, and an integral membrane A 0 domain, involved in ion translocation [2–4]. The primary struc- ture of the archaeal ATP synthase is similar to that of the eukaryotic V-ATPase, but its function as an ATP synthase is more similar to that of the F-ATP synthas- es. ATP is synthesized or hydrolysed on the A 1 head- piece, consisting of an A 3 :B 3 domain, and the energy provided or released during this process is transmitted to the membrane-bound A 0 domain. Energy coupling between the two active domains occurs via the so- called stalk part(s) [5]. Keywords A 1 A 0 ATP synthase; archaeal ATPase; F 1 F 0 ATP synthase; nuclear magnetic resonance; V 1 V 0 ATPase Correspondence G. Gru ¨ ber, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore Fax: +65 6791 3856 Tel: +65 6316 2989 E-mail: ggrueber@ntu.edu.sg (Received 25 September 2007, revised 4 February 2008, accepted 14 February 2008) doi:10.1111/j.1742-4658.2008.06338.x The boomerang-like H subunit of A 1 A 0 ATP synthase forms one of the peripheral stalks connecting the A 1 and A 0 sections. Structural analyses of the N-terminal part (H1–47) of subunit H of the A 1 A 0 ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectros- copy. Our initial NMR structural calculations for H1–47 indicate that amino acid residues 7–44 fold into a single a-helical structure. Using the purified N- (E1–100) and C-terminal domains (E101–206) of subunit E, NMR titration experiments revealed that the N-terminal residues Met1–6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1–47 interact specifically with the N-terminal domain E1–100 of subunit E. A more detailed picture regarding the residues of E1–100 involved in this association was obtained by titration studies using the N-terminal peptides E1–20, E21–40 and E41–60. These data indicate that the N-terminal tail E41–60 interacts with the N-terminal amino acids of H1–47, and this has been confirmed by fluo- rescence correlation spectroscopy results. Analysis of 1 H– 15 N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101–206 show no obvious interaction between the C-terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of sub- units E and H, and their arrangement within A 1 A 0 ATP synthase. Abbreviations FCS, fluorescence correlation spectroscopy; HSQC, heteronuclear single quantum coherence; NTA, nitrilotriacetic acid. FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS 1803 Low-resolution structures of the enzyme show that the A 1 ATPase is rather elongated, with an A 3 :B 3 headpiece and an elongated stalk [6], composed of the subunits C, D and F [6–10]. Two- and three- dimensional reconstructions of the entire A 1 A 0 ATP synthase, obtained by single-particle analysis of nega- tively stained molecules, revealed novel structural fea- tures such as two peripheral stalks and a collar-like structure [10,11], which have been proposed to com- prise the subunits H, I and E, respectively, [9,10,12]. Recently, a high-resolution structure of subunit E (residues 81–198) of the A-ATP synthase from Pyro- coccus horikoshii OT3 has been reported, showing that the dimeric C-terminal domain of subunit E con- sists of four antiparallel b-strands and six a-helices [13]. Most recently, the boomerang-like shape of sub- unit H in solution has been described for the A 1 A 0 ATP synthase from Methanocaldococcus jannaschii [12]. In these studies, a subtractive approach using truncated variations of H (H8–104, H1–98, H8–98 and H1–47) was used to understand the contributions of termini to the overall structure of subunit H and the orientation of the peripheral stalk within the enzyme. Here we describe structural studies on the N-termi- nal part of subunit H, H1–47, of the A 1 A 0 ATP syn- thase from M. jannaschii in solution using NMR spectroscopy. Two-dimensional 1 H– 15 N heteronuclear single quantum correlation (HSQC) spectra provided a unique opportunity to analyse the interaction between H1–47 and the N- and C-terminal domains of the pro- posed neighbouring subunit E. Results Resonance assignments for the N-terminal domain of subunit H (H1–47) A crucial step in identifying the residues involved in protein–protein interactions is the process of sequen- tial assignment of amino acids. Sequential assignment of H1–47 was performed using a combination of triple-resonance backbone experiments [HNCACB, CBCA(CO)NH] and 3D 15 N-resolved [ 1 H, 1 H] NOESY. Assignments of the resolved backbone resi- dues of H1–47 are presented on a 2D 1 H– 15 N HSQC spectrum (Fig. 1). The Ca chemical shift devi- ation from the random coil values (D 13 Ca) was used to predict the secondary structure of H1–47 [14]. The predicted fold consists of a single helix in the middle of the protein, with some flexible residues (Met1–6 and Leu44–Cys47) at both termini as shown in Fig. 2. Expression and purification of the N- (E1–100) and C-terminal (E101–206) domains of subunit E The full-length E subunit of A 1 A 0 ATP synthase from M. jannaschii is composed of 206 amino acids, divided into a predicted a-helix at the N-terminal part (amino acids 1–100) and an a-helical and b-sheet-containing domain at the C-terminal part (residues 101–206) [13]. For the structural studies, two truncated forms of subunit E, E1–100 and E101–206, were generated. SDS–PAGE of the recombinant E1–100 and Fig. 1. Two-dimensional 1 H– 15 N-HSQC spectrum of H1–47 in 25 m M sodium phos- phate buffer (pH 6.5) at 15 °C. Backbone and amide assignments (Asn and Gln) are shown for each residue. The HSQC cross- peak for the side chain of residue R29 is folded in the 15 N dimension and indicated by ‘R29sc’. Signals from side-chain NH 2 groups are connected by horizontal lines. Assignment of subunits E and H in A-ATP synthase S. Gayen et al. 1804 FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS E101–206 revealed prominent bands of about 12 kDa for both proteins, which were found entirely within the soluble fraction. A Ni 2+ –nitrilotriacetic acid (NTA) resin column and an imidazole gradient (10– 300 mm) in buffers 1 and 2 were used to separate subunits E1–100 and E101–206, from the main con- taminating proteins. E1–100 or E101–206 eluting at 100–300 mm imidazole were collected and subse- quently applied to an ion-exchanger column. Analysis of the isolated proteins by SDS–PAGE revealed the high purity of the truncated subunits (see supplemen- tary Fig. S1A,B). MALDI-MS showed that the dehy- drated proteins E1–100 and E101–206 have molecular masses of 11317.48 and 11837.69 Da, respectively, confirming the sequence-based predicted mass. Size- exclusion chromatography (see Experimental proce- dures) revealed that the hydrated protein spanning residues 101–206 was produced as a soluble dimer, as confirmed by solution X-ray scattering experi- ments in which molecular masses of 21.8 ± 1.5 (E1–100) and 22.5 ± 1.0 kDa (E101–206) were deter- mined (A. M. Balakrishna & G. Gru ¨ ber, unpub- lished results). The secondary structure of both proteins was determined from CD spectra measured between 185–260 nm (see supplementary Fig. S1A,B). The minima at 222 and 208 nm and the maximum at 192 nm indicate the presence of a-helical structures in E1–100. The secondary structure content of this con- struct was calculated to be 71 ± 2% a-helix and 21 ± 2% random coil (see supplementary Fig. S1A). The overall spectrum is in agreement with the pre- dicted secondary structure of the protein based on its amino acid sequence. The ratio (h 222 ⁄ h 208 )of molar ellipticity values at 222 and 208 nm was calcu- lated to be 0.95, indicating that a-helical regions within E1–100 are closely packed and are involved in a-helical interactions. E101–206 comprises 51 ± 2% a-helix and 28 ± 2% b-sheet (see supplementary Fig. S1B). Interactions of H1–47 and the N-terminal domain E1–100 studied by NMR Recently, the dimer formation of the H1–47 form has been demonstrated using small-angle X-ray scattering experiments, in which a molecular mass of 12.5 ± 2 kDa was determined for H1–47 [12]. In our work, NMR titration experiments using 1 H– 15 N HSQC spec- tra were used to characterize the interactions between subunit H1–47 and the two dimeric domains E1–100 and E101–206, respectively. Two sets of titrations were performed: HSQC spectra of 15 N-labelled H1–47 were recorded in the absence or presence of increasing amounts of unlabelled E1–100 and E101–206 separately. Figure 3A shows sections from the overlaid 2D 1 H– 15 N HSQC spectra of the H1–47 domain (shown in blue) and the H1–47–E1–100 complex (shown in red), highlighting differences in terms of changes in chemical shift fo r several r esidues. In addition, the entire H SQC spectrum of H1–47 in the absence and presence of E1–100 is shown in supplementary Fig. S2, with an inset showing the concentration-dependent increase in chemi- cal shift changes of the residue Met1. The combined ( 1 H ⁄ 15 N) chemical shift perturbations are shown in Fig. 4. A number of residues show s ignificant chemical shift perturbations upon binding of 15 N-labelled H1–47 to E1–100. There is a chemical shift perturbation in the N-terminal region of the primary sequence of H1–47 (resi- dues 1–6, 10, 11, 15, 20 and 24). By comparison, when an equimolar amount of t h e C-terminal domain of subunit E, E101–206, was added to H1–47, no significant change in the spectrum could be detected (see supplemen- tary Fig. S3). In order to map the region of E1–100 involved in the interaction with H1–47, the latter was titrated with the peptides 1MKLMGVDKIKSKILDDA KAE20 (E1–20), 21ANKIISEAEAEKAKILEKAK40 (E21–40) and 41EEAEKRKAEILKKGEKEAEM60 (E41–60). The HSQC spectrum of 15 N-labelled H1–47 in the presence of peptide E41–60 shows chemical shift Fig. 2. The amino acid sequence of H1–47 and the secondary structure elements based on 13 Ca chemical shifts with respect to the random coil values. S. Gayen et al. Assignment of subunits E and H in A-ATP synthase FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS 1805 changes for the N-terminal amino acids (Fig. 3B) that are similar to those observed in the presence of the entire E1–100 domain (Fig. 3A). In contrast, no change in the spectra were observed in NMR experiments in which 15 N-labelled H1–47 was supplemented with the peptides E1–20 or E21–40 (see supplementary Fig. S4A,B). Binding of H1–47 to peptide E41–60 studied by fluorescence correlation spectroscopy To confirm the H1–47 ⁄ E41–60 binding, fluorescence correlation spectroscopy (FCS) was used, in which E41–60 was labelled with Atto488. As a reference, a mean count rate of 33.4 kHz was determined for Atto488–E41–60. Fitting of the autocorrelation func- tions resulted in a characteristic diffusion time of 47.2 ls for the Atto488-labelled subunit E41–60. Figure 5A shows the autocorrelation curves for the fluorescent-labelled peptide E41–60 in the absence and presence of H1–47. The addition of H1–47 caused a significant change in the mean diffusion time t D , which increased with increasing concentrations of H1–47. The increase in the diffusion time was due to the increase in the mass of the diffusing particle when A B C Fig. 3. Sections from the overlaid 2D 1 H– 15 N-HSQC spectra of H1–47 in the absence (red) and presence (black) of 1.5 equivalents of unlabelled E1–100 (A). Overlay of 2D 1 H– 15 N-HSQC spectra of H1–47 in the absence (red) and presence (black) of 1.5 equivalents of E41–60 peptide (B). (C) Overlay of 2D 1 H– 15 N-HSQC spectra of the F subunit in the absence (red) and presence (black) of 1.5 equivalents of unlabelled E101–206. All the spectra were collected in a Bruker Avance 600 MHz spectrometer in 25 mM sodium phosphate buffer (pH 6.5) at 15 °C. Assignment of subunits E and H in A-ATP synthase S. Gayen et al. 1806 FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS Atto488–E41–60 interacted with H1–47. A binding constant of 3.0 lm for binding of Atto488–E41–60 to H1–47 was calculated (Fig. 5B). No binding of H1–47 was observed when labelled E1–20 or E21–40 were used in the experiments. NMR titration of subunit F and the C-terminal domain E101–206 Recently, we obtained the solution structure of sub- unit F of the methanogenic A 1 A 0 ATP synthase using NMR, and showed that it has a distinct two-domain structure, with a globular N-terminus of 78 residues and a C-terminal tail comprising residues 79–101 [15]. The N-terminal domain of subunit F is close to the bottom of the rotary D subunit [16], which is in close proximity to the C-terminal part of subunit E [9]. Based on these results, we examined the possibility of interaction between subunit F and E101–206 using NMR titration experiments. Assignments of the resolved backbone residues of subunit F are shown on a2D 1 H– 15 N HSQC spectrum (Fig. 3C) [16]. When E101–206 was titrated to the labelled subunit F, no sig- nificant chemical shift changes were observed, indicat- ing that there is no interaction between the proteins. Discussion Previous fitting of the X-ray coordinates of the atomic models of subunit A from P. horikoshii [17] and subunit B of Methanosarcina mazei Go ¨ 1 [18] into the electron density map of the A-ATP synthase from Thermus thermophilus [11], obtained from single-parti- cle analysis of negatively stained electron micrographs, allowed clear orientation of the three A subunits inside the map, thereby highlighting the position of the so- called ‘non-homologous region’ of subunit A [17]. This region of subunit A, an insert of 80–90 amino acids, which is similar to the catalytic A subunits in the related eukaryotic V-ATPases, can be crosslinked via the peptide Thr106–Arg122 to the C-terminal peptide Ile74–Lys80 of subunit H in the complete A 1 A 0 ATP synthase; this crosslinking is dependent on nucleotide binding to the catalytic site of subunit A [9]. Quantita- tive titration of subunit H to the catalytic A subunit showed that subunit H binds in a saturable fashion to subunit A with a K d of 206 nm [12]. Determination of the shape of this hydrated subunit H in solution using small-angle X-ray scattering showed that the protein is an elongated dimer with a boomerang-like shape, divided into two arms that are 12.0 and 6.8 nm long [12]. CD spectra of the protein indicated that sub- unit H has a high helical content (78%) and a high 1.0 A B 0.8 0.6 0.4 0.2 Normalized autocorrelation functionRel. bound fraction (%) 0.0 100 50 0 1E-6 1E-5 1E-4 1E-3 Correlation time (s) Concentration of H1–47 (µ M) 0.01 0.1 1 10 100 0.01 0.1 Fig. 5. H1–47 ⁄ E41–60 binding studied by fluorescence correlation spectroscopy. (A) Normalized autocorrelation functions of Atto488– E41–60 obtained by increasing the quantity of the H1–47 domain (from left to right: 0 n M,50nM, 0.1, 2.0, 7.0 and 50 lM). (B) Con- centration-dependent binding of peptide E41–60 to the H1–47 domain. The binding constant was calculated using the two-compo- nent fitting model of the CONFOCOR 3 software. The best fit to the binding constants is shown as a non-linear, asymptotic fit. Fig. 4. Combined amide ( 1 H) and nitrogen ( 15 N) chemical shift changes ([(D 1 H N ) 2 +(D 15 N p.p.m. ⁄ 6.51) 2 ] 0.5 ) for H1–47 and E1–100 binding as a function of the amino acid sequence. S. Gayen et al. Assignment of subunits E and H in A-ATP synthase FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS 1807 degree of coiling. Together with the high yield of disulfide formation of an N-terminal truncated protein, H1–47, containing a Glu47Cys mutation, it has been suggested that the helices inside the dimer of sub- unit H are in a parallel and in-register arrangement [12]. Secondary structure prediction of H1–47 based on chemical shift indices [14], using Ca and Ha chemi- cal shifts with respect to random coil values, and anal- ysis of NOESY data, confirms the high a-helix content comprising residues Glu7 to Lys43 (Fig. 2). Compari- son of the shape of subunit H and the C-terminal trun- cated form H1–98, derived from small-angle X-ray scattering data, allowed assignment of subunit H to the peripheral stalk in the two-dimensional projection of A-ATP synthase [12]. The second peripheral stalk of the A 1 A 0 ATP synthase (as shown in Fig. 6B) is predicted to be formed by subunit a [9]. Connected via its C-terminal arm to the catalytic A subunit, sub- unit H exceeds the total length of the A 1 headpiece and the central stalk [6,10] and becomes oriented with its N-terminal arm close to the collar-like structure of the enzyme complex, predicted to be formed by subunit E [9,12]. Recently, an E–H complex has been described, using electrophoresis and mass spectrometry [13]. In the NMR titration and FCS experiments pre- sented, we show that it is the N-terminal domain (E1–100, E41–60) of subunit E that specifically binds to the very N-terminus of H1–47. The high a-helical content of E1–100 (71%) might indicate that the amino acid region E41–60 (41EEAEKRKAEILKKG EKEAEM60) of the E1–100 domain binds to the N-terminal residues 1–6, 10, 11, 15, 20 and 24 of H1–47 via a helix–helix interaction. In contrast, no binding was observed with the C-terminal form, E101–206. The crystallographic structure of the C-terminal half of subunit E (E81–198) from P. hori- koshii OT3 consists of six a-helices and four antiparal- lel b-strands that form a dimer [13]. These results are comparable to the CD data obtained here for the C-terminal part, E101–206, of the M. jannaschii pro- tein (51 ± 2% a-helix and 28 ± 2% b-sheet) and the apparent size of the hydrated E101–206 based on AB Fig. 6. Topological model of the subunits in the methanogenic A 1 A 0 ATP synthase. (A) Pyrococcus horikoshii A-ATP synthase subunit A (orange, pdb 1vdz [17]), Methanosarcina mazei Go ¨ 1 A-ATP synthase subunit B (green, pdb 2c61 [18]), the bovine mitochondrial F-ATP syn- thase c subunit (violet; pdb 1e1q, chain G [30]), which is homologous to subunit D of the A-ATP synthase, and M. mazei Go ¨ 1 A-ATP syn- thase subunit F (red, pdb 20V6 [15]) were fitted into the electron density map of the A 1 ATPase [7] and A 1 A 0 ATP synthase [18], obtained from single-particle analysis electron micrographs [11]. The figure was prepared using PyMOL (http://www.pymol.org). (B) The arrangement of subunits in the A 1 A 0 ATP synthase. One B subunit has been removed from the A 1 section to reveal the D subunit within the A 3 B 3 hexamer. The A subunit is attached to the N-terminal domain (E N ) of subunit E by the peripheral stalk subunit H, and the C-terminal part of subunit E (E C ) is in close proximity to the coupling subunit D. Asterisks indicate some of the crosslinks that have been generated to probe the function and location of these subunits [9]. Assignment of subunits E and H in A-ATP synthase S. Gayen et al. 1808 FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS exclusion chromatography. Previous crosslinking experiments with the methanogenic A 1 A 0 ATP synthase showed that subunits D and E can crosslink readily via the peptides 127LDEAAKK134 and 119AYS- SKESEELVK130, respectively [9]. The homologous peptide E81–198 in the P. horikoshii OT3 structure forms the second helix (a2), which is exposed to the solvent [13], allowing crosslinking to occur between the C-terminal domain of subunit E and the central stalk subunit D (Fig. 6B). There is also biochemical evidence that subunit F of the A-ATP synthase is in close contact with subunit D [8,15]. As demonstrated by the solution structure, the four-stranded b-sheet in the N-terminal part of sub- unit F forms a hydrophobic surface, which is suggested to mediate the interaction of both subunits (Fig. 6A) [15]. Such positioning of subunit D relative to sub- unit F might bring the latter in close proximity to the C-terminal domain of subunit E. However, the data presented show no obvious interaction between sub- unit F and E101–206, indicating that subunit E mainly interacts with subunits H and D via its N- and C-ter- minal parts, respectively. In summary, the data presented support the view that the a-helical subunit H forms one of the two peripheral stalks of the enzyme, with its C-terminus connected to the N-terminal part of the catalytic A subunit and its N-terminus in close contact with the N-terminus of subunit E, with the latter being in close connection via its C-terminus to subunit D. The nucleo- tide-dependent crosslink formation between subunits A and H, the close proximity of subunit H via its N-terminus to subunit E, and the proximity of sub- units D and E leads us to speculate whether both subunits H and E might be involved in coupling and ⁄ or regulatory events in the A-ATP synthase. Experimental procedures Materials ProofStartÔ DNA polymerase and Ni 2+ –NTA chromato- graphy resin were obtained from Qiagen (Hilden, Ger- many); restriction enzymes were purchased from Fermentas (St Leon-Rot, Germany). Chemicals for gel electrophoresis were obtained from Serva (Heidelberg, Germany). BSA was purchased from GERBU Biochemicals (Heidelberg, Ger- many). Atto488–maleimide was obtained from ATTO-TEC (Siegen, Germany). All other chemicals were at least of analytical grade and were purchased from BIOMOL (Ham- burg, Germany), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), Sigma (Deisenhofen, Germany) or Serva (Heidelberg, Germany). ( 15 NH 4 ) 2 SO 4 and ( 13 C) glucose were purchased from Cambridge Isotope Laborato- ries (Andover, MA, USA). Expression, production and purification of proteins In order to amplify the two truncated constructs of sub- unit E (E1–100 and E101–206), the primers 5¢-GTTG CCA TGGCTGTGAAATTGATGGGA-3¢ (forward), 5¢-CTCCG AGCTCTCATGGCAGTTTAAC-3¢ (reverse) and 5¢-ATA CCATGGAACAGCCAGAGTATAAAG-3¢ (forward), 5 ¢- AGG GAGCTCTCAGAATAACTTCTCTGTA-3¢ (reverse), respectively, were designed (restriction sites are underlined). Genomic DNA from M. jannaschii ATCCÒ 43067DÔ was used as the template. Following digestion with NcoI and SacI, the PCR products were ligated into pET9d1-His 3 using T4 DNA ligase (the reaction mixture was incubated at room temperature for 1 h). The insert-containing pET9d- His 3 vector was transformed into Escherichia coli cells (strain NovaBlue) by electroporation (using 2500 V voltage, 25 lF capacitance and 200 W resistance), and transformants were selected on Luria–Bertoni (LB) agar plates containing 30 lgÆmL )1 kanamycin and 12.5 lgÆmL )1 tetracyclin. The cloned vector was isolated using a QIAquickÒ miniprep kit (Qiagen) and transformed into E. coli cells (strain BL21) by electroporation. The liquid cultures were shaken at 200 r.p.m. in 30 lgÆmL )1 kanamycin-containing LB medium for about 20 h at 30 °C. Production of proteins E1–100 and E101–206 was induced when the attenuance at 600 nm (D 600 ) reached 0.6 using a final concentration of 1 mm iso- propyl-b-d-thiogalactopyranoside. Following a 4 h induc- tion in a shaker at 200 r.p.m. and 30 °C, the cells were harvested at 7000 g for 15 min at 4 °C. Subsequently, cells were lysed on ice by sonication for 3 · 1 min in buffer 1 (50 mm Tris ⁄ HCl, pH 7.5, 200 mm NaCl, 1 mm phenyl- methanesulfonyl fluoride and 0.8 m m dithiothreitol for E1– 100 and E101–206, respectively) and 3 · 1 min in buffer 2 (50 mm Hepes, pH 7.0, 150 mm NaCl, 1 m m phenylmethane- sulfonyl fluoride and 0.8 mm dithiothreitol). The lysate was incubated in a waterbath for 20 min at 70 °C, and solu- ble proteins were separated from the cell debris by centrifu- gation at 10 000 g for 35 min. The supernatant was filtered (0.45 lm; Millipore, Billerica, MA, USA) and passed over a Ni 2+ –NTA resin column to isolate E1–100 and E101–206, according to the method decribed by Gru ¨ ber et al. [19]. The His-tagged protein was allowed to bind to the matrix for 1.5 h at 4 ° C and eluted using an imidazole gradient (10–300 mm) in buffer 1 for E1–100 and in buffer 2 for E101–206. Fractions containing E1–100 were identified by SDS–PAGE [20], pooled and applied to an ion exchanger (MonoQ HR5 ⁄ 5, GE Healthcare, Singapore) equilibrated using buffer A (50 mm Tris ⁄ HCl, pH 7.5, 1 mm phenyl- methanesulfonyl fluoride, 1.0 mm dithiothreitol). The pro- tein was eluted using a linear gradient with buffer B (50 mm S. Gayen et al. Assignment of subunits E and H in A-ATP synthase FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS 1809 Tris ⁄ HCl, pH 7.5, 1 m NaCl, 1 mm phenylmethanesulfonyl fluoride, 1.0 mm dithiothreitol) at 3 mLÆmin )1 . In the case of E101–206, the protein was further purified using ResourceQ (6 mL, GE Healthcare) as the ion-exchanger col- umn and equilibrated in buffer C (50 mm Hepes, pH 7.0, 1mm phenylmethanesulfonyl fluoride, 1.0 mm dithiothrei- tol). The protein was then eluted using a linear gradient with buffer D (50 mm Hepes, pH 7.0, 1 m NaCl, 1 mm phenyl- methanesulfonyl fluoride, 1.0 mm dithiothreitol). The pro- teins were concentrated as required using Centricon YM-3 spin concentrators (Millipore) with a 3 kDa molecular mass cut-off. Subunit F and the truncated form of subunit H, H1–47, respectively, were isolated as described previously [9,12]. For production of uniformly labelled ( 15 N and 15 N ⁄ 13 C) subunit F and H1–47, the bacteria were grown in M9 mini- mal medium containing 15 NH 4 Cl or 15 NH 4 Cl ⁄ ( 13 C)glucose. The purity and homogeneity of all protein samples were analysed by SDS–PAGE [20]. SDS gels were stained with Coomassie brilliant blue G250. Protein concentrations were determined using a bicinchoninic acid assay (Pierce, Rockford, IL, USA). Size-exclusion chromatography Size-exclusion chromatography was performed on a Super- dex 75 10 ⁄ 30 column (GE Healthcare) at 0.5 mLÆmin )1 using 50 mm Hepes, pH 7.0, 150 mm NaCl and 1 mm dithiothrei- tol. The elution profiles were recorded by determining the A 280 values. The molecular masses of E1–100 and E101–206 were estimated by comparison with the 25 kDa (chymo- trypsinogen A) and 13.7 kDa (RNase A) markers of the GE Healthcare low-molecular-weight gel filtration calibra- tion kit. CD spectroscopy Steady-state CD spectra were obtained in far-UV light (185–260 nm) using a CHIRASCAN spectropolarimeter (Applied Photophysics, Leatherhead, UK). Spectra were collected in a 60 lL quartz cell (Hellma, Mu ¨ llheim, Ger- many) with a path length of 0.1 mm, at 20 °C and with a step resolution of 1 nm. The readings were for an average of 2 s at each wavelength, and the recorded ellipticity val- ues were the mean of three determinations for each sample. CD spectroscopy of the two proteins (2.0 mgÆmL )1 ) was performed in a buffer of 50 mm Tris ⁄ HCl, pH 7.5, 200 mm NaCl, 1 mm dithiothreitol for E1–100 and of 50 mm Hepes, pH 7.0, 150 mm NaCl, 1 mm dithiothreitol for E101–206. The spectrum for the buffer was subtracted from the spectrum of the protein. CD values were converted to mean residue ellipticity (h, degree cm 2 Ædmol )1 ) using chira- scan software version 1.2 (Applied Photophysics). This baseline-corrected spectrum was used as the input for com- puter methods to obtain predictions of secondary structure. In order to analyse the CD spectrum, the following algorithms were used: VARSELEC [21], Selcon [22], Contin [23] and K2D [24] (all methods incorporated into the pro- grams dicroprot [25] and neuralnet [26]). NMR data collection and processing The NMR sample was prepared in 90% H 2 O ⁄ 10% D 2 O containing 25 mm NaH 2 PO 4 ⁄ Na 2 HPO 4 (pH 6.5) and 0.1% NaN 3 . All NMR experiments were performed at 15 °Con a Bruker (Rheinstetten, Germany) Avance 600 MHz spec- trometer. The experiments recorded on 15 N ⁄ 13 C-labelled samples were HNCA, HNCACB, CBCA(CO)NH and 3D 15 N-NOESY-HSQC (s m = 200 ms). Two-dimensional NOESY and TOCSY experiments were carried out using unlabelled samples. All the two- and three-dimensional experiments made use of pulsed-field gradients for coher- ence selection and artefact suppression, and utilized gradi- ent sensitivity enhancement schemes. Quadrature detection in the indirectly detected dimensions was achieved using either States ⁄ TPPI (time-proportional phase incrementa- tion) or echo ⁄ anti-echo method. Baseline corrections were applied wherever necessary. The proton chemical shift was referenced to the methyl signal of 2,2-dimethyl- 2-silapentane-5-sulfonate (Cambridge Isotope Laboratories) to 0 p.p.m. The 13 C and 15 N chemical shifts were referenced indirectly to 2,2-dimethyl-2-silapentane-5-sulfonate. All the NMR spectra were processed using either nmrPipe ⁄ nmr- Draw [27] or the in-built software topspin of the Bruker Avance spectrometer. Peak picking and data analysis for the Fourier-transformed spectra were performed using sparky [28]. NMR-binding experiments To analyse the binding between subunits H1–47 and E1–100 and between H1–47 and E101–206, a series of 1 H– 15 N HSQC spectra were recorded at 15 °C for the fixed concen- tration of 100 lm of H1–47, titrated with increasing amounts (up to 1.5 equivalents) of E1–100 and E101–206 separately. The proteins were incubated for 30 min for each step of the experiment. The change of chemical shift was monitored in the HSQC spectra. The same procedure was followed for the binding experiments with 15 N-labelled H1–47 and the N-ter- minal peptides from subunit E, E1–20, E21–40 and E41–60, as well as 15 N-labelled subunit F and E101–206. All the sam- ples used were either finally dissolved in or exchanged with 25 mm sodium phosphate (pH 6.5) buffer prior to the bind- ing experiments. Fluorescence correlation spectroscopy Fluorescence correlation spectroscopy was performed on a LSM-FCS system (confocor 3, Zeiss, Jena, Germany) Assignment of subunits E and H in A-ATP synthase S. Gayen et al. 1810 FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS using Atto488–maleimide to label peptides E1–20, E21–40 and E41–60 of subunit E. The labelling and FCS experi- ments were performed in 25 mm sodium phosphate buffer, pH 6.5, for 10 min at room temperature. The excess non- bound dye was removed five times using a ZipTipÒ P-10 pipette tip with C4 resin (Millipore), replacing the solution with 5% acetonitrile ⁄ water (0.1% trifluoroacetic acid). The sufficient removal of non-bounded dye was verified by FCS-measurements of the wash steps prior to the elution of the labelled peptide. The fluorescent-labelled peptide was subsequently eluted with 60% acetonitrile ⁄ water (0.1% trifluoroacetic acid). The 488 nm laser line of an 30 mW argon ion laser was focused into the aqueous solution using a water immersion objective (C-Apochromat 40·⁄1.2 W, korr UL-Vis-IR, Zeiss). FCS was performed on 15 lL droplets, which were placed on gelatin-treated (3% gelatin solution) Nunc 8 well-chamber cover glasses (Nunc ⁄ Denmark, catalogue number: 155411) according to the method described by Hunke et al. [29]. The following filter sets were used: MBS (main beam splitter), HFT488 (Haubtfarbteiler, main color splitter); EF (emission filter), none; DBS (dichroism beam splitter), mirror; EF2, LP505 (long pass filter). Out-of-focus fluorescence was rejected by a 90 lm pinhole in the detec- tion pathway, resulting in a confocal detection volume of approximately 0.25 fL. Fluorescence autocorrelation func- tions were measured for 30 s each with ten repetitions. Solutions of Atto488–maleimide in buffer were used as references and for calibration of the confocor 3 system. To analyse the autocorrelation functions of E41–60-bound H1–47, a standard autocorrelation two-diffusion-coefficient normalized triplet model was used for fitting (FCS-LSM software, confocor 3, Zeiss). The diffusion time for fluorescently labelled E41–60 was measured independently, and kept fixed during fitting of the FCS data. Therefore, determination of the binding constants only required calcu- lation of the relative amounts of free labelled peptide E41– 60 with the short diffusion time, in comparison with an increase of the diffusion time. The increase of the diffusion time is caused by the increment of the size of the particles because of the interaction of E41–60 with H1–47 according to the Stoker–Einstein relation. The calculations were per- formed using confocor 3 software version 4.2, Microsoft excel 2003 and origin 7.5 (Origin Lab, Northampton, MA, USA). Peptide synthesis The N-terminal peptides E1–20, E21–40 and E41–60 of M. jannaschii subunit E were synthesized and purified by RP-HPLC at the Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, Singapore. The purity and identity of the peptides were confirmed by HPLC and ESI-MS. Acknowledgements We thank Dr Subramanian Vivekanandan for his sup- port in the analysis of NMR data and critical reading of the manuscript. We are grateful to Dr C. F. Liu for synthesizing the peptides and Dr S. K. Sze for mass spectrometry analysis. S. Gayen is grateful to Nanyang Technological University for the award of a research scholarship. This research was supported by A*STAR Biomedical Research Council grant 06 ⁄ 1⁄ 22 ⁄ 19 ⁄ 467. References 1 Scha ¨ fer G, Engelhard M & Mu ¨ ller V (1999) Bioenerget- ics of the archaea. Mol Biol Rev 63, 570–620. 2 Weber J & Senior AE (2003) ATP synthesis driven by proton transport in F 1 F 0 -ATP synthase. FEBS Lett 545, 61–70. 3 Lolkema JS, Chaban Y & Boekema EJ (2003) Subunit composition, structure, and distribution of bacterial V-type ATPase. J Bioenerg Biomembr 35, 323–336. 4 Cross RL & Mu ¨ ller V (2004) The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H+ ⁄ ATP coupling ratio. 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Protein Eng 5, 191–195. 27 Delaglio GS, Vuister GW, Zhu G, Pfeifer J & Bax A (1995) NMRPipe: a multidimensional spectral process- ing system based on UNIX pipes. J Biomol NMR 6, 277–293. 28 Kneller DG & Goddard TD (1997) SPARKY 3.105 Edition. University of California, San Francisco, CA. (http://www.cgl.ucsf.edu/home/sparky). 29 Hunke C, Chen WJ, Scha ¨ fer HJ & Gru ¨ ber G (2007) Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the V 1 V 0 ATPase from Aedes albopictus. Protein Expr Purif 53, 378–383. 30 Gibbons C, Montgomery MG, Leslie AG & Walker JE (2000) The structure of the central stalk in bovine F(1)- ATPase at 2.4 A ˚ resolution. Nat Struct Biol 7, 1055– 1061. Supplementary material The following supplementary material is available online: Fig. S1. Far-UV CD spectrum of the E1–100 and E101–206 proteins, and SDS–PAGE gel showing a sample of the purified proteins. Fig. S2. Overlay of 2D 1 H– 15 N-HSQC spectra of H1–47 in the absence and presence of 1.5 equivalents of unlabelled E1–100. Fig. S3. 1 H– 15 N-HSQC spectra of H1–47 and a 1 : 1 molar mixture with E101–206. Fig. S4. 1 H– 15 N-HSQC spectra of H1–47 in the absence and presence of 1.5 equivalents of E1–20 or E21–40 peptides. in 25 mm sodium phosphate buffer (pH 6.5) at 288 K. This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article. Assignment of subunits E and H in A-ATP synthase S. Gayen et al. 1812 FEBS Journal 275 (2008) 1803–1812 ª 2008 The Authors Journal compilation ª 2008 FEBS . time, in comparison with an increase of the diffusion time. The increase of the diffusion time is caused by the increment of the size of the particles because of the interaction of E4 1–60 with H1 –47. constantly changing, and therefore need to acquire energy. The molecule ATP is the common energy currency of cells; when cells require energy, they ‘spend’ ATP. In the case of the archaea, the A 1 A 0 ATP. The arrangement of subunits in the A 1 A 0 ATP synthase. One B subunit has been removed from the A 1 section to reveal the D subunit within the A 3 B 3 hexamer. The A subunit is attached to the