Tài liệu Báo cáo khoa học: Rotary F1-ATPase Is the C-terminus of subunit c fixed or mobile? docx

Rotary F 1 -ATPase Is the C-terminus of subunit c fixed or mobile? Martin Mu¨ ller, Karin Gumbiowski, Dmitry A. Cherepanov, Stephanie Winkler, Wolfgang Junge, Siegfried Engelbrecht and Oliver Pa¨ nke Universita ¨ t Osnabru ¨ ck, FB Biologie/Chemie, Abt. Biophysik, Osnabru ¨ ck, Germany F-ATP synthase synthesizes ATP at the expense of ion motiveforcebyarotarycouplingmechanism.Acentral shaft, subunit c, functionally connects the ion-d riven rotary motor, F O , with the rotary chemical reactor, F 1 .Using polarized spectrophotometry we have demonstrated previ- ously the functional rotation of the C-terminal a-helical portion of c in the supposed ‘hydrophobic bearing’ formed by the (ab) 3 hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between the a-helix of c and subunit a did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a ‘functional unwinding’ of the a-helix to form a swivel joint. Furthermore, they suggested a firm clamping of that part of c even without the engineered cross-link, i.e. in the wild-type enzyme. He re, we rechecked the rotational mobility of the C-terminal portion of c relative to (ab) 3 . Non-fluorescent, engineered F 1 (aP280C/cA285C) was oxidized to form a (nonfluorescent) ac heterodimer. In a second mutant, containing just the point mutation within a, all subunits were labelled with a fluorescent d ye. Following disassembly and reassembly of the combined preparations and cystine reduction, the enzyme was exposed to ATP or 5¢-adenylyl- imidodiphosphate (AMP-PNP). After reoxidation, we found fluorescent ac dimers in all cases in accordance with rotary motion of the entire c subunit under these conditions. Molecular dynamics simulations covering a time range of nanoseconds therefore do not necessarily account for mo- tional freedom in microseconds. The rotation of c within hours is compatible with the spectroscopically detected blockade of rotation in the AMP-PNP-inhibited enzyme in the time-range of seconds. Keywords: ATP hydrolysis; catalytic mechanism; F 1 -ATP- ase; molecular dynamics calculation; motor protein. F O F 1 -ATP synthase of bacteria, chloroplasts, and mito- chondria catalyses the endergonic synthesis of adenosine triphosphate (ATP) from adenosine d iphosphate (ADP) and phosphate (P i ) using a transmembrane proton-motive or sodium-motive force. In reverse, F O F 1 is capable of generating ion-motive force at the expense of ATP hydro- lysis. The enzyme, in its simplest bacterial form (Escherichia coli), consists of eight different subunits, a 3 b 3 cde in F 1 ,the catalytic headpiece, and ab 2 c 10 in F O , the ion-translocating membrane portion. Energy is mechanically transferred between F O and F 1 by rotation of the central shaft (cec 10 ), relative to the stator subunits (a 3 b 3 dab 2 ). Both complexes, F O and F 1 , are rotary steppers (for recent reviews, see [1–8]). Based upon crystal structure analysis it has been hypo- thesized [9] and later s hown by chemical cross-linking [10], by polarized absorption recovery after photobleaching [11], and most spectacularly by videomicroscopy [12–14], that ATP hydrolysis by isolated and immobilized F 1 -ATPase drives the rotation of the central shaft, subunit c,relativeto the hexagon formed by subunits (ab) 3 . Portions of subunits a and b provide a snug fit for the a-helical C-terminal portion of c, considered to form a ‘hydrophobic bearing’ and to be essential for rotary function [9]. The functional rotation of the penultimate amino acid at the C-terminus of c relative to the immobilized remainder of chloroplast F 1 has b een detected by polarized photobleaching (with eosin as probe) [11,15–17]. This finding was difficult to reconcile with the observation that up to 12 amino acid residues could be deleted by site-directed mutagenesis without suppressing catalysis [18,19] or impairing c rotation [18] (Fig. 1). It was even more difficult to reconcile with the lack of inhibition of ATP h ydrolysis a nd c rotation after covalent disulfide- bridging subunits a and c at positions aP280C and cA285C [20] (Fig. 1). One way to interpret this finding was to assume that the a-helix at the C-terminal portion of subunit c was unwound to provide swivel joints a round one or several dihedral angles, in other words, that c under these conditions did not rotate in its entirety, but just in part. Molecular dynamics simulations of ac cross-linked enzyme revealed that the torque generated by the enzyme is sufficient to u nwind the a-helix at the C-terminal portion of c thus impelling the backbone rotation around Rama- chandran dihedral angles [20]. Further calculations with the noncross-linked enzyme suggested a firm clamping of the C-terminal c portion within (ab) 3 (this work). This would make the proposed unwinding of the a-helix in c afeatureof the wild-type enzyme and an integral element of the catalytic mechanism. Such a permanent immobilization of the Correspondence to O. Pa ¨ nke, Universita ¨ t Osnabru ¨ ck, FB Biologie/ Chemie, Abt. Biophysik, Barbarastr.11, D-49076 Osnabru ¨ ck, Germany. Fax: +49 541 969 2870, E-mail: opaenke@uos.de Abbreviations:F O , ion-driven rotary motor of F-ATP synthase; F 1 , rotary chemical reactor of F-ATP synthase; AMP-PNP, 5¢-adenylyl- imidodiphosphate; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); TMR-ITC, tetramethyl rhodamine-5-isothiocyanate. (Received 30 April 2004, revised 30 July 2004, accepted 6 August 2004) Eur. J. Biochem. 271, 3914–3922 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04328.x C-terminal portion of c, however, would contradict previ- ously pu blished spectroscopic results revealing its ATP- dependent functional rotation [11,17]. We reassessed the rotational mobility of this portio n of c with cleavable cross-links similar to t he approach used b y Duncan et al. [10]. The experimental design is shown in Fig. 2. By dissociation and reconstitution of appropriately tailored F 1 complexes fluorescent a subunits were incor- porated into the two noncross-linked positions of subunit a of the oxidized mutant MM10 (Fig. 2). After reduction of the nonfluorescent cross-linked ac the effects of ligand binding and catalysis on the ability of the c-C-terminus to reposition itself relative to specific a subunits was tested. Upon reoxidation we found fluorescent ac dimers after catalytic turnover or substrate binding, and even if the enzyme was left without nucleotides and phosphate. The formation o f a fluorescent ac cross-link could b e p revented only by omitting the red uction/reoxidation cycles alto- gether. T hese data reveal ed the C-terminal portion of subunit c always t o be (rotary) mobile at the time scale of this experiment, i.e. within hours. Experimental procedures Chemicals and enzymes All restriction and DNA modifying enzymes were obtained from New England Biolabs (Frankfurt/Main, Germany) or Fig. 1. Schematic re pres entation o f E. coli F 1 and the calculated unwinding of subunit c. (A) Localization of the engineered cysteine residues within E. coli F 1 -mutant MM10. Two copies each of subunits a and b are omitted for clarity. Subunit a and b areontheleftandthe right side, respectively. Both point mutations, aP280C and cA285C, are shown in black. The 12 amino acid residues shown in spacefill representation can be truncated without inhibition of the rotary mechanism [18]. Mutant MM6 has o ne cysteine, aP280C, only. The residue coordinates were from a homology model constru cted previ- ously [ 46]. (B ) Snapshots of c conformation during the f orced molecular dynamics calculated with the torque of 56 pNÆnm. The secondary structure of c is shown for the time of 1 ns (the end of initial equilibration), 17 ns (half of turnover), 23 n s (one turnover) and 32 ns (the end of final equilibration). Fig. 2. Experimental flow-chart to test the rotational motion of the C-terminal portion of subunit c. Su bunits a, b and c are shown as circles, squares and triangles, respectively. Subunits shown in grey or white are labelled or unlabelled, respec tively. Hatch ed subu nits are either labelled or not as a result of the reassociation proc ess (see text for details). Small black dots indicate the engineered cysteines aP280C and cA285C. Ó FEBS 2004 Rotation of subunit c in E. coli F 1 -ATPase (Eur. J. Biochem. 271) 3915 MBI Fermentas (St. Leon -Rot, Germany). Benzonase was from Merck (Darmstadt, Germany). Oligonucleotide primers were s ynthesized by MWG-Biotech (Ebersberg, Germany). Nickel-nitrilotriacetic acid s uperflow was ob- tained from Qiagen (Hilden, Germany) and tetramethyl- rhodamine-5-isothiocyanate (TMR-ITC) was from Molecular Probes ( Leiden, the Netherlands). All other reagents used were of the highest grade c ommercially available. Strains and plasmids The plasmids pMM10 (aP280C/cA285C) and pMM6 (aP280C) were generated as described by Gumbiowski et al. [20]. In both cases plasmid pKH7 (all wild-type cysteines substituted by alanine [21], His 6 -tag extension at the N-terminus of subunit b, cK108C [13]) was used as starting material. I n brief, the mutation cA285C was generated by standard PCR with pKH7 as template DNA and using KpnIandSacI for transferring the PCR product into pKH7 (resulting in plasmid pMM9). The mutation aP280C was generated using a method described by Weiner et al. [22] with the subclone pMM3 [pBluescript II SK(+) containing the KpnI/XhoI fragment of pKH7] as template DNA. The exchange of the KpnI/XhoI fragment o f pKH7 with the corresponding fragment carrying the aP280C mutation resulted in plasmid pMM6. Plasmid pMM10 w as generated by replacement of the KpnI/SacI fragment of pMM6 with the corresponding fragment of pMM9. E. coli strains used were DH5a for plasmid preparation and DK8, which contains a D(uncB-uncC) deletion [ 23], for expression of E. coli F 1 . Expression and purification of E. coli F 1 Preparation of F 1 was performed essentially as described previously [18] except for the following modifications. Cells were now h arvested at A 600  1.8. Furth ermore the buffer for resuspension of the cells a fter harvesting contained no EDTA-free protease inhibitor mixture tablet. Instead, the resuspended cells were incubated with ‡ 37 5 U Benzonase per 100 mL for 15–20 min at room temperature before Ribi press passage (Ribi Cell Fractionator, Model RF-1, Sorvall, Langenselbold, Germany). After elution of F 1 from the anion exchange column the solution was supplemented with 1m M MgATP and 2 m M dithiothreitol. Next, the protein was p recipitated with 3.2 M (NH 4 ) 2 SO 4 andstoredat4°C. The yield was 2.5–3.0 mg protein per litre of culture volume. Cross-linking and labelling of E. coli F 1 For cross-linking of F 1 mutant MM10 (aP280C/cA285C)  15 mg protein were purified from (NH 4 ) 2 SO 4 and dithiothreitol by gel fi ltration through PD-10 columns (Amersham Biosciences, Freiburg, Germany), which were equilibrated with 5 0 m M Tris/HCl, 50 m M KCl, 5 m M MgCl 2 , 10% (v/v) glycerol, pH 7.5 (buffer A). The eluate was supplemented with 2 m M ATP and 100 l M 5,5¢- dithiobis(2-nitrobenzoic acid) (DTNB) and the samples were incubated for 16 h at room temperature. The reaction was stopped by addition of 20 m M N-ethylmaleimide followed b y a 10 min i ncubation at room temperature. The probe was purified by gel filtration through PD-10 columns, which were equilibrated with dissociation buffer (50 m M Mes/NaOH, 1 M LiCl, 5 m M ATP, 0.5 m M EDTA, pH 6.1). For labelling of the F 1 mutant MM6 (aP280C) with the fluorescent dye TMR-ITC  30 mg protein were purified from (NH 4 ) 2 SO 4 and dithiothreitol by gel filtration through PD-10 columns, which were equilibrated w ith 100 m M HEPES/NaOH, 50 m M KCl, 5 m M MgCl 2 , pH 8.5. After determination of the protein concentration a 50-fold molar excess of TMR-ITC was added and then incubated for 1 h at room temperature. Free dye was removed by gel filtration through P D-10 columns with dissociation buffer. The de gree of labelling was d etermined by measuring the absorbance of the purified sample at the absorbance maximum of TMR-ITC (k max ¼ 555 nm, e ¼ 65000 cm )1 Æ M )1 ). The degree of labelling was usually between 20 and 30 fluorescent dyes per F 1 -MM6. Dissociation of E. coli F 1 mutants and reconstitution of hybrid-F 1 Dissociation and reconstitution of F 1 was performed essentially as described previously [10,24]. After oxidation of F 1 -MM10 and dye labelling of F 1 -MM6, the two F 1 mutants were gel filtrated against dissociation buffer (see above), m ixed in a ratio of 1 : 2 ( 10 mg F 1 -MM10 and  20 mg F 1 -MM6) and frozen in liquid nitrogen. The samples were thawed at room temperature and again frozen inliquidnitrogen,andthenstoredat)80 °C. After thawing at room temperature the dissociated samples were diluted to 0.5 mgÆmL )1 protein concentration with reconstitution buffer [50 m M Mes/NaOH, 10% (v/v) glycerol, pH 6.0] and then dialyzed (SpectraPor, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) against reconstitution buffer containing 2.5 m M MgATP for 16 h at room temperature. Reduction and reoxidation of hybrid-F 1 Reconstituted hybrid-F 1 was purified by nickel-nitrilotri- acetic acid affinity chromatography. Columns were equil- ibrated with reconstitution buffer containing 2.5 m M MgATP and bound product was washed with buffer A containing 20 m M imidazole. After elution of purified hybrid-F 1 with buffer A containing 150 m M imidazole (yielding  1.5 mg of protein per 3 mL eluate) the degree of labelling was determined as described above. Typical values for the degree of labe lling were 1–4 fluorescent dyes per hybrid-F 1 . Aliquots of reconstituted F 1 samples were treated either w ith no nucleotide, with 4 m M 5¢-adenylyl- imidodiphosphate (AMP-PNP), with 4 m M AMP-PNP and 4m M ADP, or with 4 m M ATP. The samples were then reduced by addition of 20 m M dithiothreitol and incubation for 16 h at room temperature. After another additio n of 20 m M dithiothreitol and incubation for further 2 h the samples were purified by gel filtration through NAP-10 columns (Amersham Biosc iences) and nucleotides were added as d escribed above. The f ollowing gel filtration buffers were used: (a) buffer A for the sample, which contained no nucleotide and the sample, which contained 4m M ATP, (b) buffer A + 1 m M AMP-PNP for the sample, which contained 4 m M AMP-PNP and (c) buffer A 3916 M. Mu ¨ ller et al.(Eur. J. Biochem. 271) Ó FEBS 2004 +1m M AMP-PNP and 1 m M ADP for the sample, which contained 4 m M AMP-PNP and 4 m M ADP. The samples were incubated for 2 h at room temperature and reoxidized by a two-fold successive addition of 100 l M DTNB followed by incubation at room temperature for 16 h and 2 h , respectively. The reaction was stopped by addition of 20 m M N-ethylmaleimide and incubation for 10 min at room temperature. Samples were purified by gel filtration through NAP-10 columns, which were equilibrated with buffer A. After each reaction/purification step aliquots were taken for determination of ATP hydrolysis activity, protein concentration and for SDS/PAGE. Molecular dynamics calculations A three-dimensional model of the ‘hydrophobic b earing’ at the C-terminal portion of c was built using the X-ray structure of bovine enzyme (PDB entry 1E79 [25]). The rotary shaft is comprised of 24 residues from the N-end of c subunit (cA1–cK24) and 43 residues from its C-end (cT230– cL272). The chosen portion included a major part of the coiled-coil region of c and the complete a-helical C-terminus as held within the top of (ab) 3 . The rotary axis z was aligned along the main axis of the shaft. The ‘bearing’ included a total of 138 residues from the neighbouring portion of (ab) 3 located within 1.8 nm from the rotary axis (70 residues belonged to a and 68 residues to b). Hydrogen atoms and terminal groups of the protein backbone w ere built by the program CHARMM 22 [26]. The protein was ‘solvated’ by TIP3 rigid water molecules [27], which formed a cylinder with diameter of 3.6 nm and height of 7.6 nm. In total, the system contained 2952 protein atoms a nd 1104 water molecules. The molecular dynamics simulations were per- formed with the program NAMD 2 [28] using the all-atom empirical force field CHARMM 22 [26]. A harmonic boundary potential was applied to prevent water evaporation outside the cylinder considered above. The backbone atoms of the ‘bearing’ were constrained at their crystallographic posi- tions, while other protein atoms were unconstrained. The electrostatic interactio ns w ere truncated by a cut-off distance of 1.2 nm. The s ystem was equilibrated during 1 n s, and then the rotation of c was forced by a constant torque applied t o the coiled-coil portion of c at the level of cK18–cK21 and cD233–cS236 residues. The t orque was created by external forces acting on the two groups of four carbon atoms each. The first group included the C a atoms of cK18, cI19, cT20 and cK21, and the second group C a atoms of cD233, cN234, cA235 and cS236. The magnitude and direction of the forces was calculated at every step of the molecular dynamics integration (1 fs step width) by a Tcl script (http://www.tcl.tk) using the current position of the geometrical center of each group relative to the z-axis. Ab initio quantum chemistry calculations These calculations were carried out within the limits o f the ab initio Hartree–Fock method in the 6 –311++G basis set using the GAMESS program complex [29]. The model s ystem included a n A la-Gly dipeptide in t he neutral state with an amidated C-terminus. The equilibrium configuration of this dipeptide was obtained by the geometrical optimization in the molecular mechanics force field, followed by semi- empirical AM1 minimization , and finall y b y ab initio minimization in the 6–311++G basis set. The potential energy profiles along w and / dihedral coordinates were calculated by the rotation of the dipeptide in discrete equidistant 15° steps with the subsequent complete geom- etry opt imization i n t he 6–311++G basis at the fixed values for w or /, respectively. The potential energy of the optimized structure was calculated in the 6–311++G basis set using the second-order Mo ¨ ller–Plesset c onfiguration- based correlation method [30]. Other procedures ATP hydrolysis activity w as measured by determination of released P i after incubation of the enzyme for 5 min at 37 °C in a reaction mixture containing 50 m M Tris/HCl, 3 m M MgCl 2 ,10m M NaATP, pH 8.0. The blue-coloured phos- phomolybdate complex was photometrically detected at a wavelength of 745 nm [31]. SDS/PAGE was carried out in the Amersham Biosciences Phast system (Amersham Bio- sciences) without 2-mercaptoethanol in the sample buffer. Gels were stained with Coomassie Brilliant Blue R-250 [32] and s ilver [33]. P rotein determinations were carried out according to the method of Sedmak & Grossberg [34]. Results In the two E. coli F 1 mutants, MM10 and MM6, u sed in this study, all wild-type c ysteines were substituted b y alanines, one novel cysteine in c (K108C) was introduced and a His 6 -tag at the N-terminus of subunit b was added [13]. MM10 contained t wo additional cysteines in positions aP280C and cA285C, and was capable of forming a cross- link upon oxidation with a yield of more than 98% [20]. Mutant MM6 contained only one additional cysteine in position aP280C. In E. coli strain DK8 both mutants grew on succinate as well as t he control (KH7 [13]). After isolation and purification, ATPase activities under r educing conditions were in the range of 130–160 UÆmg )1 for both mutants, without noticeable amounts of c ross-linked ac (Fig. 3 , lanes 1 & 3). Figure 2 summarizes the protocol used to test the rotational mobility of the C-terminal portion of subunit c. Cross-link formation and labelling Mutant MM10 showed f ormation of an ac heterodimer upon oxidation with DTNB. After 16 h incubation, th e c monomer had disap peared completely, as checked by SDS/ PAGE(Fig.3,lane1&2).MM6failedtodoso,as expected (Fig. 3, lane 3 & 4). Despite the cross-link MM10 showed normal ATP hydrolysis activities and c rota tion [20]. This was previously interpreted such that the torque generated b y ATP hydrolysis is sufficient to uncoil the a-helix in the C-terminal portion of subunit c [20]. MM10 served as source for nonfluorescent ac hetero- dimers. For the incorporation of fluorescent a subunits into the two nonfluorescent a positions within F 1 ,mutantMM6 was labelled with the amine-reactive fluorescent dye TMR- ITC. Conditions were chosen to ensure a labelling by 20–30 TMR molecules per molecule of MM6. As shown in Fig. 3, lane 5, the l abelling affected all five F 1 subunits. An Ó FEBS 2004 Rotation of subunit c in E. coli F 1 -ATPase (Eur. J. Biochem. 271) 3917 additional band of high molecular mass as apparent in the SDS gel was shown by Western blotting to consist of a subunits only, but not c [20]; probably an aa homodimer. It should be noted that MM6 could not form an ac heterodimer because it lacked the essential c ysteine residue (cA285C;Fig.3,lane4). Reconstitution of hybrid-F 1 Labelled MM6 and cross-linked MM10 were dissociated by a freeze-thaw proc edure in the presence of 1 M LiCl according to [ 10,24]. The sample s were m ixed at a ratio of 2 : 1 (MM6/MM10), dissociated, diluted and dialyzed against reconstitution buffer containing 2.5 m M MgATP. By application of nickel-nitrilotriacetic acid affinity chro- matography, we obtained a solution containing a mixture of labelled hybrid-F 1 along with unknown amounts of His 6 - tagged b.Startingwith30mgofF 1 , about 1.5 mg protein were obtained from the nickel-nitrilotriacetic acid column, i.e. 5%. Assuming a homogeneous hybrid-F 1 preparation, labelling ratios of 1–4 fluorescent dye molecules per F 1 were determined. Two types of hybrid-F 1 species were expected, depending on the origin o f c. O ne population of F 1 complexes was expected to contain nonfluorescent ac heterodimers originating f rom mutant M M10, whereas the second type should contain fluorescently labelled c from MM6. Both types were expected to contain both fluorescent and nonfluorescent subunits a and b (Fig. 2 ). The latter type was unimportant in this context, as these enzymes lacked the capability to form fluore scent ac cross-links, due to the absence of the point mutation cA285C. Figure 3, lane 6, shows t he result of the SDS/PAGE of the hybrid-F 1 preparation. The absence of c monomers in the SDS gel indicated that the first type of hybrid-F 1 molecules, containing nonfluorescent ac heterodimers, was formed exclusively during the reconstitution procedure. The reason for the absence of hybrid-F 1 containing fluorescent subunit c from MM6 is unknown. Possibly, attached TMR molecules prevented the formation of reassembled e nzymes due to steric hindrance. Fluorescent a subunits were present in hybrid-F 1 molecules, as was evident from the fluorescence image of the SDS gel (Fig. 3, lane 6). The ab band, as well as the aa homodimer band, were fluorescent. The activities of hybrid-F 1 were dependent on the resulting labelling ratio. Preparations with labelling ratios of about one dye molecule per protein molecule had ac tivities of 110 UÆmg )1 ,which were close to the original activities of the mutants MM10 and MM6 (130–160 UÆmg )1 ). At ratios of about four the activity was about 40 UÆmg )1 . This decrease, however, was probably not only caused by the fluorescent dye, but also by the presence of nonfunctional reassembled enzyme and single b subunits. Rotational mobility of the C-terminal portion of subunit c Hybrid-F 1 , which contained the nonfluorescen t ac cross- link, was expected to reveal fluorescent ac heterodimers after reduction of the disulfide bridge, followed by rotation of c upon ATP hydrolysis and subsequent reformation of the disulfide bridge. To this end, aliquots of reconstituted F 1 samples were exposed to (a) no nucleotide at all, (b) AMP- PNP, (c) AMP-PNP and ADP, or (d) ATP. Samples w ere reduced by addition of dithiothreitol followed by gel filtration in the presence of the respective substrate. Afterwards, the disulfide bridge was r eformed by addition of DTNB. After each reaction/purification step s amples were taken for determination of ATP hydrolysis activity and SDS/PAGE. Table 1 summarizes the activities of a ll samples. In order to compare the values fro m different expe riments with different labe lling ratios the activities were normalized with respect to the activity of the primary nickel-nitrilotriacetic acid eluate. The relative activities remained unchanged during the whole reduction/reoxidation procedure. The high activity of the oxidized s amples and the quantitative c ross- linking of subunit c with a (SDSgelinFig.3,lanes 8,10,12,14) suggested the unwinding of the a-helix of subunit c in hybrid-F 1 molecules, as seen before with MM10 [20]. The lack of inhibition by AMP-PNP i s Fig. 3. SDS/PAGE (A) and the corresponding fluorescence images (B). An 8–25% gradient gel (Amersham Biosystems Phast system) with 2% (w/v) SDS was used and stained with Coomassie Brilliant Blue R-250 [32] followed by silver [33]. The protein concentration was 3 mgÆmL )1 ; each lane con tained 0 .9 lg protein. Lanes 1–5 show the starting material, F 1 mutants MM10 and MM6, in the reduced and oxidized state as indicated. MM6 in lane 5 was labelled with 24 dye molecules. Lane 6 was the hybrid-F 1 preparation before substrate incubation. Lanes 7 –14 show the reduced and reoxidized e nzyme under di fferent substrate conditions, as indicated. T he nucleotide concentration was 4m M for all nucleotides, throughout. Lane 15 shows hybrid-F 1 ,which was handled like the other samples but w as never reduced (control). The labelling ratio of hybrid-F 1 was 1–4 TMR m olecules per F 1 . 3918 M. Mu ¨ ller et al.(Eur. J. Biochem. 271) Ó FEBS 2004 understandable, because the samples were diluted during the activity a ssay and a dded ATP displaced the residual amounts of AMP-PNP and ADP from the catalytic sites of the enzyme. Activity measurements in the presence of 1 m M AMP-PNP or a mixture of 1 m M AMP-PNP and 1 m M ADP showed complete inhibition. Figure 3 shows the corresponding SDS/PAGE analysis ofthesamplesaftereachreactionstepandTable2 summarizes the fluorescence intensities of the correspond- ing g el bands. After reduction of the hybrid-F 1 preparation a c monomer band became clearly visible and a minor amount of ac heterodimers was not reduced (Fig. 3 , lanes 7,9,11,13). As expected, the re oxidation of the samples intensified the ac bands again and the c bands disappeared completely (lanes 8,1 0,12,14). At the same t ime the ac heterodimers showed fluorescence, consistent with a rota- tional movement of the C-terminal portion of subunit c. This behaviour was independent of the applied substrate conditions. Even with AMP-PNP and a mixture of AMP- PNP and ADP a fluorescent ac band was observed. This was surprising because the ATP analogue AMP-PNP is known to stabilize F 1 complexes and was added to crystallization media in X-ray structure analysis [9,35–38]. To exclude that the fluorescent ac heterodimer was formed due to impurities (e.g. nonreconstituted subunits) a control sample was treated like the other samples with respect to incubation times, gel filtration, etc., but without being reduced. This c ontrol sample s howed a strong ac ban d in the SDS gel, but no fluorescence (Fig. 3, l ane 15). We checked whether or not a fl uoresc ent ac dimer was formed after reduction due to a continuous disassembly/assembly mechanism of F 1 molecules. For this purpose we labelled wild-type-F 1 enzymes (BWU13 [39]), with TMR-ITC and mixed them with an unlabelled F 1 mutant, KH7, carrying a His 6 -tag at the N-terminus of subunit b [13]. After overnight incubation, both mutants were separated by nickel-nitrilotriacetic acid chromatography. The eluted His 6 -tagged F 1 remained nonfluorescent ( < 3%), thus excluding any interchange of subunits. Nevertheless, it was apparent that the fluorescence intensity of the ac bands in all reoxidized samples was rather weak, a lthough the SDS band was very intense. This was not surprising, because not all i nserted a and b subunits were labelled and only a maximum of two-thirds of all ac heterodimers could have contained a fluorescent a subunit. In fact, our results show that about 14% of the total intensity was located in the ac band (Table 2). Molecular dynamics simulations of the rotary mobility of c within the ‘hydrophobic bearing’ at the top of a 3 b 3 The molecular model of the rotary part of c and the surrounding part of (ab) 3 was constructed as described in Experimental procedures using available model coordinates [9]. Unlike p revious simulations with the a-helical terminus of subunit c from E. coli, the present simulations included a large portion of the ‘hydrophobic bearing’ at the top of a 3 b 3 and the rotation of c was restricted only by steric interactions with this hydrophobic collar. The system was equilibrated for 1 n s, after that a rotary motion of c was forced by a constant torque applied to its coiled-coil portion at the l evel of cK18–cK21 and cD233–cS236. The simula- tions included two traces obtained with the applied torque of 56 and 112 pNÆnm, respectively (the average torque generated by the enzyme at physiological conditions has been found to be as high as 56 p NÆnm [40]). The angular displacement of c as a function of time (calculated at the level where the torque was applied) is shown in Fig. 4. The right curve in this figure was obtained with a torque of 56 pNÆnm and the left one with a torque of 112 pNÆnm, the arrows show the beginning (t ¼ 1ns)andtheend(t¼ 23 ns) of the forced rotation. With 56 pNÆnm torque, the relaxation of the system was calculated during the last 8 ns of the dynamics. In both cases the a pplied torque caused a complete unfolding of the single-helical portion of c at the level of cR254–cV257 residues, a partial deformation of the double-helical part of c, but the C-terminal portion of c was tightly clamped within (ab) 3 and k ept its initial c onforma- tion and orientation. Table 1. Normalized activities of hybrid-F 1 preparations. After recon- stitution a nd purification the hybrid-F 1 preparations had activities between 40 and 110 UÆmg )1 , depending on the resulting labelling ratios (dye/protein), which had values between 4 and 1, respectively. In order to compare different experiments with different dye contents the activities were normalized to 100 with respect to the activity of the primary nickel-nitrilotriacetic acid eluate. Substrate for incubation None ATP AMP-PNP AMP-PNP +ADP Nickel-nitrilotriacetic acid eluate 100 100 100 100 Reduced 120 149 102 112 Reoxidized 113 134 119 104 Table 2. Fluorescence intensities of the SDS gel bands. The fluorescence shown in Fig. 3 was analyzed with the GELPRO ANALYSER software from Media Cybernetics (Silver Spring, MD, USA). The band intensities were baseline corrected and normalized to 100 with respect to the total intensity of all bands in each sample lane. Red, reduced; Reox, reoxidized: Ox, oxidized. Band Substrate for incubation None ATP AMP-PNP AMP-PNP + ADP Control Red Reox Red Reox Red Reox Red Reox Ox a 2 22 54 23 3 320 ac 2 17 2 12 3 12 2 14 2 ab 84 73 81 80 79 70 90 79 73 Ó FEBS 2004 Rotation of subunit c in E. coli F 1 -ATPase (Eur. J. Biochem. 271) 3919 With 56 pNÆnm torque, the secondary structure was stable during the first 10 ns, after that a partial unfolding began by a rotation of the peptide backbone between cA256 and cV257 residues. The initial conformational transition included a simultaneous shift of the Ramachandran angle w of cA256 by +120° and of the angle / of cV257 by )90°. After t his initial unfolding event, fur ther unfolding occurred, mainly due to rotation around w angles of cR254, cA256, cQ255 and cV257 residues. At the end of forced dynamics, the angle w of cA256 made a full turnover by +360°. When the external torque was switched off at t ¼ 23 ns, the warped double-helical part of c underwent an elastic relaxation back to its initial outstretched conforma- tion, whereas the conformation of cR254–cV257 residues remained uncoiled. When a twofold higher torque of 112 pNÆnm was applied, the secondary structure a-helix was broken after 2.5 n s of the forced dynamics, the initial unfolding event included almost simultaneous changes of Ramachandran angles w of cR254 and cA256 by +120° and angles / of cQ255 and cV257 by )90°. The further rotation of c was caused mainly by rotation around w-angles of cR252, cT253, cA256 and cV257 residues. The residues cR252 and cT253 made more than one turnover around w-angle. In both cases the m olecular dynamics simulations revealed that rotation around the w Ramachandran angle w as preferred over that around /. We calculated the potential barrier f or the rotation around w and / angles in the dipeptide Ala-Gly by ab initio quantum chemistry (program GAMESS [29] using Pople’s 6–311++G basis set and the second-order Mo ¨ ller–Plesset configuration-based correla- tion method). The potential barriers for the rotation a long the wand /dihedral angles were 30 and 3 8 kJÆmol )1 , respect- ively. These values were about 25% higher than the figures obtained by the molecular mechanics calculations [20]. The calculations indicated that in the crystallographic structure the C-terminal portion of c seems to b e tightly clamped within the ‘hydrophobic bearing’ at the top of (ab ) 3 . The steric constraints on the c rotation in this region were essentially bigger than the rigidity of the single a-helix. The secondary structure o f t he latter c ould be e asily unfolded when the operational torque of 56 pN Ænm was applied to the rotary shaft. At this magnitude of torque the rotation around Ramachandran angles in the region o f residues cT253–cV257 (cA267–cS271 in E. coli F 1 [41]) proceeded with a rate of 10 8 s )1 , four orders of magnitude faster than the observed r otary transitions in the enzyme [42]. Because the molecular dynamics simulations were performed with the frozen tertiary conformation of (ab) 3 ,it remained unclear whether large-scale fluctuations of the (ab ) 3 structure can open the ‘bearing’ at the time scale o f ls to ms that is required for a rotation of c as a whole within (ab ) 3. Discussion This study was motivated by the previous finding that a mutant F 1 (MM10) with a disulfide bridge engineered between the stator subunit a and the C-terminal portion of the rotary shaft, subunit c, showed unimpaired ATPase activity and full torque in the videomicroscopy assay for rotation [20]. The robustness of the bearing collar of (ab) 3 and the rotational shaft, c, has been demonstrated by other approaches: up to 12 amino re sidues at t he C-terminal portion of subunit c were dispensable for catalysis and rotation [18,19]. The C-terminus could be extended by 16 amino acid residues without drastic consequences (a frameshift acco mpanied by b suppressor mutations) [43]. Gre en fluo rescent p rotein could be fused to the C-terminus of c without loss of enzyme function [44]. The crystal structure clearly pointed to limited freedom of c to rotate other than around its long (‘vertical’) axis (original suggestion by Abrahams et al.[9])andinthe ‘hydrophobic bearing’ formed by subunits a and b around the C-terminal portion of c. Molecular dynamics calcula- tions ([20] and this work) suggested the unwinding of the single a-helix at the C-terminal portion of c, thus allowing for unimpaired rotation of the remainder of c.Furthermore, the calculations suggested the very end of c to be clamped within the N-terminal collar of subunits ( ab) 3 permanently and even without a disulfide bridge (this work), in seeming contradiction with previous work employing the polarized photobleaching of eosin [11,17]. The d ata presented here are clearly indicative of a movement of the C-terminal portion of subunit c relative to (ab ) 3 within the time domain investigated, because the originally cross-linked ac heterodimer consisted only of nonfluorescent polypeptides, whereas after reduction/reoxi- dation the r espective band contained fluorescent a.More- over, the appearance of the fluorescent b and was not dependent on conditions allowing for ATP hydrolysis. The protocol we used did not (as with the original one [10]) allow discrimination between translational o r rota- tional movement of c. Because the microvideographic data [12], however, clearly demonstrated unidirectional c rota- tion upon ATP hydrolysis (even in the millisecond time Fig. 4. Forced ro tation of c within (ab) 3 . The molecular model included the central a-helical shaft (it covered the 3.2 nm long single-helical and the 3.2 nm long double-coiled portions of c) and 138 residues of (ab) 3 located within a 7.6 nm long cylinder at the distance up to 1.8 nm from the rotation axis. The constant torque of 56 and 112 pNÆnm (the steeper and flatter curves, respectively) was applied to the two groups of four C a atoms located on e turn abo ve the low er end o f c.The arrows indicate the time interval of forced dynamics, after that the system relaxation was monitored during 8 ns. 3920 M. Mu ¨ ller et al.(Eur. J. Biochem. 271) Ó FEBS 2004 range [42]), we interpret our findings also as indicative for rotation, not just translation, even though s ome d ata reported in the literature are still not compatible with the concept of rotational catalysis [45]. Whether t he same holds true for t he AMP-PNP experi- ment is more difficult to decide. Compared to the results of Duncan et al. [10], where the corresponding disulfide bridge was located close to the DELSEED sequence (bD380C/ cC87), we found fluorescent ac heterodimers even without catalytic activity and in the p resence of AMP-PNP. T his finding is in contrast with our previous observation b y polarized photobleaching [11,17], which revealed blockage of the functional rotation of c in some milliseconds by added AMP-PNP, but it agrees well with the dat a of Duncan et al.[10],wherec was allowed to rotate for about 10 min. In the presence of ATP, they observed an increased amount of radiolabelled bc dimers, compatible with c rotation. The yield of radioactive bc was decreased to about 30% upon inhibition or in the a bsence of ATP, but not to zero (Figs 3 and 4 in [ 10]). In other words, i n t hese experiments also, movement of c could not be blocked entirely over a long time span. Movement of c was probably possible within the time scale of hours dictated in our approach by the required protocol, as AMP-PNP might have been dissociated and rebound occasionally. Whether c under these conditions was able to carry out full rotation remains an open question, at least its rotational o r translational freedom sufficed to allow interaction with another a subunit than the one it was connected to originally. An inspection of the X-ray struc- ture, however, raises serious doubts about whether just a ‘bending’ movement of this portion of c might occur at all and also at the same time be sufficient to induce the observed cross-link. A rotational movement, this time perhaps only around 120° and without preferential direc- tion, thus would seem more plausible. Why has the molecular dynamics calculation produced a different result? Ignoring the limited section of the enz yme that entered into the calculations and the fixed backbone at the N-terminal portion of a and b, a simulation covering some nanoseconds still cannot acco unt for domain flexibil- ity in the range of microseconds. Evidence for such fluctuations was obtained in our previous studies. The rotational relaxation of the eosin attached to the C-terminal portion of chloroplast c had components in the nanosecond time range, but also another one at 30 ls (Fig. 4 in [17]). These components have been interpreted to reveal the librational motion of the dye molecule in narrow constraints (ns) and subsequently in wider constraints by fluctuations (30 ls) o f the N-terminal collar of (ab) 3 . The different methodological approaches, the time domains they apply to, and the associated mobility of subunit c are summarized in Fig. 5. The emerging picture is that the central shaft of F 1 never comes to a full halt; c is free to slowly rotate back and forth in the time range of minutes to hours, owing to the exchange of bound substrates or inhibitors. ATP hydrolysis, on the other hand, causes rapid unidirectional rotation in millisec- onds and predominates so that futile mobility escapes detection in activity a ssays. Solely in the time range of nanoseconds the C-terminal portion might be permanently clamped as proposed by the molecular dynamics calcula- tions. A remarkable exception is the cross-linked mutant MM10, where ATP hydrolysis-induced rotation overcomes the artificial clamping of the C-terminal portion probably by unwinding the a-helix to form a swivel joint. Acknowledgements Skillful technical assistance by Gabriele Hikade and Hella Kenneweg is gratefully acknowledged. This work was supported by grants from the DFG (SFB 431/P1) to W.J. and S.E., by the HSFP to W .J., by the Volkswagenstiftung to W.J. and O.P., and the Fonds der Chemischen Industrie to W.J. References 1. Boyer, P.D. (1997) The ATP synthase – a splendid molecular machine. Annu.Rev.Biochem.66, 717–749. 2. Capaldi, R.A. & Aggeler, R. (2002) Mechanism of the F(1)F(0)- type ATP synthase, a biological rotary m otor. Trends Biochem. Sci. 27, 154–160. 3. Fillingame, R.H. & Dmitriev, O.Y. (2002) Structural model of the transmembrane F o rotary sector of H + -transporting ATP syn - thase derived by solution NMR and intersubunit cross-linking in situ. Biochim. Biophys. Acta 1565, 232–245. 4. Junge, W., Pa ¨ nke, O., Cherepanov, D., Gumbiowski, K., Mu ¨ ller, M. & Engelbrecht, S. (2001) Inter-subunit rotation and elastic power transmission in F o F 1 -ATPase. 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The secondary structure of c is shown for the time of 1 ns (the end of initial equilibration),. o the coiled-coil portion of c at the level of cK18–cK21 and cD233–cS236 residues. The t orque was created by external forces acting on the two groups of