Rotor architecture in the yeast and bovine F1-c-ring complexes of F-ATP synthase

Journal of Structural Biology 177 (2012) 490–497

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Rotor architecture in the yeast and bovine F1-c-ring complexes of F-ATP synthase Marie-France Giraud a,b,⇑, Patrick Paumard a,b, Corinne Sanchez a,b, Daniel Brèthes a,b, Jean Velours a,b, Alain Dautant a,b,⇑ a b

Univ. de Bordeaux, IBGC, UMR 5095, F-33000 Bordeaux, France IBGC CNRS, UMR 5095, F-33000 Bordeaux, France

a r t i c l e

i n f o

Article history: Received 22 June 2011 Received in revised form 7 October 2011 Accepted 27 October 2011 Available online 18 November 2011 Keywords: Crystal structure Molecular motor F1FO-ATP synthase

a b s t r a c t The F1FO-ATP synthase is a rotary molecular nanomotor. F1 is a chemical motor driven by ATP hydrolysis while FO is an electrical motor driven by the proton flow. The two stepping motors are mechanically coupled through a common rotary shaft. Up to now, the three available crystal structures of the F1c10 subcomplex of the yeast F1FO-ATP synthase were isomorphous and then named yF1c10(I). In this crystal form, significant interactions of the c10-ring with the F1-head of neighboring molecules affected the overall conformation of the F1-c-ring complex. The symmetry axis of the F1-head and the inertia axis of the c-ring were tilted near the interface between the F1-central stalk and the c-ring rotor, resulting in an unbalanced machine. We have solved a new crystal form of the F1c10 complex, named yF1c10(II), inhibited by adenylyl-imidodiphosphate (AMP-PNP) and dicyclohexylcarbodiimide (DCCD), at 6.5 Å resolution in which the crystal packing has a weaker influence over the conformation of the F1-c-ring complex. yF1c10(II) provides a model of a more efficient generator. yF1c10(II) and bovine bF1c8 structures share a common rotor architecture with the inertia center of the F1-stator close to the rotor axis. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction In the first bovine F1-ATPase (bF1) crystal structures, the central stalk was disordered (Abrahams et al., 1994; Stock et al., 2000). The structure of bF1 inhibited by DCCD was the first model with a fully resolved central stalk (Gibbons et al., 2000). Afterward, progressive dehydration of bF1 crystals that induced unit-cell shrinkage and increased symmetry-related contacts has improved the resolution from 6.5 to 3.8 Å (Sanchez-Weatherby et al., 2009) and 1.9 Å (Bowler et al., 2007). The reduction of the crystal cell along the maximum dimension of the molecule is concomitant with the twist of the lower part of the central stalk (Bowler et al., 2006a,b, 2007). The compliance of the central stalk under crystal-packing forces supports its intrinsic flexibility. In the yeast F1-ATPase (yF1) crystal structure (Kabaleeswaran et al., 2006), the foot of the central stalk of two independent copies are stacked foot-to-foot and were relatively well defined while that of the third copy, which Abbreviations: yF1c10(I), crystal form I of yeast F1c10 sub-complex; yF1c10(II), crystal form II of yeast F1c10 sub-complex; yF1, yeast F1-ATPase structure; bF1c8, crystal of bovine F1c8 sub-complex; TMH, transmembrane helix; EM, electron microscopy; AMP-PNP, adenylyl-imidodiphosphate; DCCD, dicyclohexylcarbodiimide; F1-statorT, bovine F1-OSCP-b99-214-d1-118-F6; BDFsol, bovine b79-183-d3-123-F65-70. ⇑ Corresponding authors. Address: IBGC CNRS, 1 rue Camille Saint-Saëns, 33077 Bordeaux cedex, France. Fax: +33 556 999 051. E-mail addresses: [email protected] (M.-F. Giraud), a.dautant@ ibgc.cnrs.fr (A. Dautant). 1047-8477/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2011.10.015

was not involved in crystal contacts, was fully disordered. The yeast central stalk appeared more twisted relative to the bovine ones. The first crystal structure of F1c10 sub-complex (yF1c10) of the yeast F1FO-ATP synthase established the stoichiometry of the subunit c in the c-ring (Stock et al., 1999). Crystals of the F1c10 subcomplex of the yeast F1FO-ATP synthase inhibited by ADP and likely azide (yF1c10:ADP) belong to P21 monoclinic space group and the structure was refined at 3.4 Å resolution (Dautant et al., 2010). A few months later, coordinates of the crystal structure of the yeast F1c10 inhibited by adenylyl-imidodiphosphate, yF1c10:AMP-PNP, refined at 3.0 Å resolution was deposited to the Protein Databank (Pdb Id: 2xok). These three crystals belong to the P21 monoclinic space group and were isomorphous. This crystal form will be further named yF1c10(I). The overall refined structures were very similar with a rmsd of 0.9 Å (after superposition of 99% of the total number of Ca). In the yF1c10:AMP-PNP, an AMP-PNP molecule was present in both the bDP- and bTP-sites and the aDP–bDP and aTP–bTP pairs were slightly open and very closed, respectively, corroborating the putative presence of an azide molecule at the bottom of these catalytic sites in yF1c10:ADP, as initially shown in the bovine F1:ADP:azide structure (Bowler et al., 2006a). Moreover, the crystal packing is atypical for membrane proteins (Iwata, 2003). The top of the F1-head and the bottom of the c10-ring of adjacent molecules interact to form infinite columns. The principal inertia axes of the central shaft and of the c-ring are tilted near the

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attachment of the ring to the subunit c that is consequently thrown off center. Such an inclination of F1 relative to the c10-ring posed a problem. According to this model, when the ring rotates, the top of the central stalk and thus the F1-stator will make an eccentric movement. To solve this dilemma, a hand-over-hand mechanism, by coordinating structural changes in subunits c, has been proposed (Kinosita et al., 2000). Recently, the bovine bF1c8 complex provides a more accurate model of the F1-c-ring domain in the intact bovine F1FO-ATP synthase (Watt et al., 2010). Since the subunit a and other subunits of peripheral stalk lack in the yeast and bovine F1-c-ring X-ray structures, the membrane rotor/stator interface remain unknown. However, the cryo-electron microscopy (EM) map of intact yeast F1FO-ATP synthase resolves the internal structure of the membrane region, especially the c-ring and the subunits a(6) and b(4) (Lau et al., 2008). Here, we describe a new crystal form of the yeast F1c10 subcomplex, further named yF1c10(II) in which crystal packing weakly affects the rotor conformation. Despite a low resolution, the overall fold is clearly visible with a more straight C-terminal helix of subunit c. Though the pseudo three-fold axis of the F1-stator is slightly tilted relative to the c-ring axis, its center of mass is located approximately on this axis. The present yeast yF1c10(II) and the bovine bF1c8 models (Watt et al., 2010) are comparable and provide accurate models of the F1-c-ring domain in the intact F1FO-ATP synthase.

damage (Flot et al., 2010). Appropriate data collection segments were determined using STRATEGY routine of MOSFLM (Leslie, 1992) and collected using 2.3° oscillation for a complete range of 180°. A complete data set was processed at 6.5 Å resolution using MOSFLM and scaled with SCALA (CCP4, 1994) without noticeably diffraction anisotropy. yF1c10(II) belongs to monoclinic P21 space group and has one molecule of F1c10 sub-complex in the asymmetric unit resulting in a solvent content and a Matthews coefficient of 65.3% and 3.69 Å3 Da 1, respectively. Data collection statistics are summarized in Table 1. 2.4. Dehydration of yF1c10:ADP crystals Controlled dehydration of yF1c10:ADP crystals did not result in significant changes in cell parameters, presumably because the contacts between adjacent molecules in yF1c10(I) form are highly stable along the ac-face diagonal, preventing the access to other states. This stability was confirmed by the large anisotropy of diffraction with the highest diffraction limit in the corresponding direction of the reciprocal space. Crystals are longest along the baxis and were usually mounted in the cryo-loop along this particular direction. The yF1c10(II) form appears to be more relevant for such experiments, although up to now we were unable to reproduce this crystal. 2.5. Structure solution and refinement

2. Material and methods 2.1. Enzyme purification and inhibition Mitochondria were obtained with the zymolyase method from the modified D273-10B/A/H/U yeast strain (met6, his3, ura3) harboring a deletion of the TIM11 gene and a (His)6-tag at the C-terminus of subunit i, a component of the yeast peripheral stalk. Subsequent purification steps of F1FO-ATP synthase were performed as previously described (Talbot et al., 2009; Dautant et al., 2010). The final enzyme concentration (10 mg/ml) was assessed by the Lowry method. The enzyme was incubated for 1 h 30 at 4 °C with 40 lM ADP and 1 mM AMP-PNP and then overnight at 4 °C with 100 lM DCCD and 0.02% azide. Inhibition conditions by nucleotides were similar to bF1:AMP-PNP (Abrahams et al. 1994) and yF1c10:AMP-PNP (Pdb Id: 2xok; Stock et al., 2010).

The structure of yF1c10(II) was determined by molecular replacement using the coordinates of the yF1c10(I) split in two independent parts, one includes a F1-sector and the other a c10-ring (Pdb id: 2xok, Stock et al., 2010). Molecular replacement was performed with the PHASER program (McCoy et al., 2007). Different stoichiometries of the subunit c (8, 9 and 11) of the ring gave solutions with lower Z-scores. In the initial rA-weighted 2Fo–Fc electron density maps displayed using COOT (Emsley and Cowtan, 2004), the F1 and the c10-ring clearly appears in the density (Fig. 1). In the rA-weighted Fo–Fc electron density maps, there is no density ascribable to any subunits of the peripheral stalk. A typical refinement stage consisted of 10 runs of rigid body refinement. The rigid body groups contained individual structural domains of

Table 1 X-ray data and refinement statistics.

2.2. Crystallization Crystallization was carried out at 20 °C, by the sitting drop vapor diffusion method, after mixing equal volumes of protein and reservoir solutions (100 mM NaCl, 10% PEG 4000, 3 mM sodium azide, 100 mM MES, pH 6.5). One crystal grew in a few days as a flattened rod of 200 lm in length and 50  20 lm2 in width (Fig. S1). This crystal, yF1c10(II), without visible signs of twinning was flash-frozen in liquid nitrogen, with addition of 15% (v/v) glycerol as cryoprotectant. The crystallization conditions were similar to those of yF1c10(I) and yF1c10:AMP-PNP (Pdb Id: 2xok). Otherwise, a set of yF1c10:ADP crystals was prepared as previously (Dautant et al., 2010) to carry out controlled dehydration on yF1c10(I) using the HC1b humidity-control device mounted on the ID14-1 ESRF MX beamline (Bowler et al., 2010). 2.3. Data collection and treatment X-ray diffraction data of the crystal yF1c10(II) were collected on the microfocus beamline ID23-2 (ESRF, Grenoble) to significantly reduce the background. Moreover a helical data collection mode was selected to minimize exposure to X-rays and thus radiation

Space group

P21

Unit-cell dimensions a, b, c, (Å); b, (°) Molecules per a.u. VM; (%) solvent X-ray source Wavelength, (Å) Resolution range, (Å) No of unique reflections No of free reflections Completeness, (%) Multiplicity Rmergea Bfact from Wilson plot, Å2 Rcryst/Rfreeb Root-mean-square deviations Bond lengths, (Å) Bond angles, (°) No. of atoms

107.69, 174.91, 164.26; 93.92 1 3.54; 65.31 ID23-2 beamline ESRF 0.8726 54.62–6.50 (6.85–6.50) 11,687 (1716) 559 (137) 96.6 (97.2) 3.5 (3.5) 8.9 (1.8) 0.117 (0.921) 274 0.317(0.325)/0.339(0.361) 0.003 0.944 30,100

Values in parentheses are for the highest resolution shell. P P P P a Rmerge = h i |Ii(h) |/ h i Ii(h), where Ii(h) is the ith measurement of reflection h and is a weighted mean of all measurements of h. P P b R = h |Fo(h) Fc(h)|/ h |Fo|. Rcryst and Rfree were calculated from the working and test reflection sets, respectively. The test set constituted 4.8% of the total reflections not used in refinement.

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of the membrane stator, i.e., the five TMHs of subunit a, the two Nterminal TMHs of subunit 4 and the single TMH of subunits f, 8 and i, for which no high resolution structural data are available. The yeast subunit a(6) has five predicted transmembrane helices (TMHs). A model of the aTMH2-aTMH5 was built by homology using the Escherichia coli subunit a model based on NMR data and disulfide crosslinks (Pdb ID: 1c17; Rastogi and Girvin, 1999). Then, a yeast ac10 complex was modeled using the yeast c10-ring structure (Dautant et al., 2010) and the E. coli ac12 complex model (Rastogi and Girvin, 1999). It was placed against the c10-ring at the suggested membrane rotor/stator interface, according to the interaction geometry of the E. coli ac12 complex. aTMH4 (aR176) and aTMH5 interact with two cTMH2 (cE59) of the c-ring. The five predicted transmembrane helices from subunits 4, i, f and 8 were added as ideal a-helix segment, on the basis of inter subunit disulfide bridge formation (Velours and Arselin, 2000). In yeast, the monomeric complex is devoid of dimer-specific subunits e and g (Arnold et al., 1998). The atomic coordinates and structure factors of yF1c10(II) model have been deposited in the Protein Data Bank (accession code 3zry).

3. Results and discussion 3.1. The rotor is less involved in crystal contacts in yF1c10(II) than in yF1c10(I)

Fig.1. rA weighted 2Fo–Fc electron density map at 6.5 Å resolution from final refinement of yF1c10(II) around the subunits aTP, bDP, c, d and e and the c10-ring are shown in ribbon representation and colored in blue, red, yellow, magenta, cyan and green, respectively. Electron density map is contoured at 1.0 r level. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.)

the F1-sector subunits or each a-helix of subunits c. NCS constrains were used for the c10-ring. A total of five such refinement steps were carried out using the PHENIX.REFINE program (Adams et al., 2004; Afonine et al., 2005). In the central cavity of the c10ring, an elongated residual density blob could result from disordered lipids or detergent molecules. The final Rcryst/Rfree values were 0.32/0.34, respectively (Table 1). The surface areas and hydrogen bonds were calculated using the PISA server (http:// www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver). Principal axes, shortest distances between axes and angles between vectors were calculated using EDPDB (Zhang and Matthews, 1995). Figures were made using PYMOL (DeLano, 2002).

2.6. Fitting of atomic models and docking into cryo-EM maps Atomic models of the F1-OSCP-b99-214-d1-118-F6 (F1-statorT) and b79-183-d3-123-F65-70 (BDFsol) sub-complexes of the bovine ATP synthase were superimposed using b160–183 and F68–53 residues to straighten out the bend of the peripheral stalk at residue b146 (Kane Dickson et al., 2006; Rees et al., 2009). The resulting models were superimposed with the yeast F1-c-ring using the subunits a. Finally, the hybrid atomic model of F1-c-ring-statorT was docked into yeast cryo-EM maps (Lau et al., 2008) using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (Pettersen et al., 2004). The hybrid model lacks any of the 10 predicted TMHs

In yF1c10(I) crystals, it was suspected that the position of the F1head relative to the c-ring was influenced by numerous crystal contacts (Figs. 2A, S2 A and B). Despite a limited resolution, the present yF1c10(II) crystal structure (Figs. 2B, S2 C and D) provides a means to appreciate the crystal packing effect over the overall yF1c10 sub-complex. The unit-cell dimensions of yF1c10(I) (P21; a = 135.0 Å, b = 173.9 Å, c = 137.0 Å, b = 92.7°) and yF1c10(II) (P21; a = 107.7 Å, b = 174.9 Å, c = 164.3 Å, b = 93.9°) are different. Though the b-cell dimensions are roughly equal, a 25% decrease in the acell dimension and a 20% increase in the c-cell dimension of yF1c10(II) result in a small 4% increase in the unit-cell volume. The Matthews coefficient and the solvent content are therefore of the same order of magnitude than those of yF1c10(I). At first sight, crystal packings are similar (Fig. S2). F1c10 molecules are parallel to the long diagonal of the ac-faces and form infinite columns (Fig. S2 A and C). Parallel columns form layers that are stacked anti-parallel along the monoclinic screw b-axis (Fig. S2 B and D). Nevertheless, a detailed inspection reveals minor differences but with significant consequences. In yF1c10(I), the acface is roughly square with the long ac-diagonal of 188 Å in length (Figs. 2A and S2A). Within a column, although the successive c-ring axes are 20 Å apart, the axes of the c-ring and the F1-stator of adjacent molecules converge at the contact region (Fig. 2A). At the interface, the small and narrow protrusion made by the inner helix of subunits c at the bottom of the ring of one molecule fits over the larger axial cavity of the b-crown on top of the (ab)3 assembly of an adjacent molecule, burying 980 Å2 of accessible surface area (asa). This interaction results in a bend of the rotor. In yF1c10(II), the acface is rectangular (Fig. 2B). Molecules are also aligned along the long ac-diagonal but 196 Å apart. The c-ring axes of successive molecules are 16.4 Å apart and the symmetry axes of the c-ring and the F1-stator of adjacent molecules do not converge at the contact region (Fig. 2B). Interestingly, the protrusion of the c-ring lies over the crown of the adjacent (ab)3 assembly burying only 180 Å2 of asa (Fig. 2B). Such an interface cannot significantly influence the overall conformation of the rotor. Within layers, in the yF1c10(I) ac plane, parallel columns are 96 Å apart. F1-heads of about 110 Å of diameter are inserted

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Fig.2. Crystal packing effect on the overall conformation of the F1-c-ring complex in yF1c10(I) (A) and yF1c10(II) (B). The views are seen along the b-axis. The axes of the catalytic head, D(ab)3, (blue) and of the c-ring, Dc10-ring, (red) of the same complex and of the adjacent complex along the long diagonal of the ac-face are drawn. Intramolecular (dashed arrows) and intermolecular (plain arrows) contact surface areas between F1 sector and c-ring are given in Å2. The subunits a and b are colored the same to show the 60° rotation of columns between the two crystal forms (Fig. S2). The c-rings of adjacent symmetry related molecules are colored in green. The white crosses represent the center of mass of (ab)3 heads. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.)

between c-rings with a smaller size (Ø 57 Å). So, there are no direct protein–protein contacts between parallel columns (Fig. S2A). Intra-layer assembly could involve the annular belt of disordered detergent molecules surrounding the c-rings. In yF1c10(II), parallel columns are 82 Å apart and are more nested (Fig. S2C). Molecules interact through two interfaces along the short a-axis. At one interface, the a-helical domain of the subunits aE and bE lies over the ab nucleotide binding domain of the subunits aDP and bTP (387 Å2). At the other one, the subunits de interact locally with the a-helical domain of the subunit aDP (42 Å2). Unlike in yF1c10(I), there are direct contacts in the three directions of yF1c10(II) crystal. Finally, in both yF1c10 crystal forms, inter layer contacts occur through subunits a (Fig. S2B and D). However, when both lattices are viewed down the b-axis, individual columns of yF1c10(I) are rotated by 60° along the column-axis with regard to the yF1c10(II) one. Thus, by combination with the monoclinic screw b-axis, columns of adjacent layers are related by a 120° rotation. Consequently, the subunit aE is in contact with subunit aDP (625 Å2) in yF1c10(I) instead in contact with subunit aTP (672 Å2) in yF1c10(II). Whatever the crystal form, these contacts involve the same peripheral loops (a103–108, a119–126, a189–195, a223–229 and a258–264) of the central nucleotide-binding domain of symmetry-related subunits a. The bovine F1c8 complex (bF1c8) crystallizes in the orthorhombic space group P212121 with a fully different packing (Watt et al., 2010). Molecules draw zigzag lines (Fig. S3A). The bottom of the c8-ring points perpendicularly towards the opening of the interface aE/bE of the adjacent F1-head with a contact interface of about 400 Å2 (Fig. S3A and B). A second interface of 220 Å2 (Fig. S3B) involves the subunit d and the subunit aE of another adjacent molecule whereas contacts between F1-stators are spread out over two interfaces for a total of 886 Å2 (not shown). Finally, the crystal contacts involving rotor subunits are smaller in yF1c10(II) (220 Å2, 18% of the total lattice contact area of 1280 Å2) than in bF1c8 (620 Å2, 41% of the total lattice contact area of 1500 Å2) or in yF1c10(I) (980 Å2, 61% of the total lattice contact area of 1600 Å2). To accommodate large packing crystal interfaces, proteins must undergo significant conformation changes, thus yF1c10(II) provides a more accurate model of the F1-c-ring domain in the intact yeast F1FO-ATP synthase like the bF1c8 does for the bovine enzyme (Watt et al., 2010).

3.2. The outer surfaces of yeast and bovine rotor c-rings overlap when the stator (ab)3 symmetry axes are superimposed The subunit c forms a left-handed 2-stranded canonical antiparallel coiled-coil (residues 1–31 and 225–253). In yF1c10(II), it adopts a winding state like in yF1 and yF1c10(I) structures (rmsd 0.9 Å after superimposing of 92% of the Ca of the subunit c). The coiled-coil is overwound relative to the bovine bF1c8 and bF1:DCCD subunit c (rmsd 1.4 Å after superimposing of 80% of the Ca) with the largest shifts occurring in the two ends of the long C-terminal a-helix and in the loop c189–204 (in yeast numbering) in contact with the c-ring (Fig. 3 and Table 2). The pseudo three-fold axes of the (ab)3 domains of bF1c8, yF1c10(I) and yF1c10(II) were superimposed using the subunits (Fig. 4). Then, the relative rotation of the central stalk is defined by the angular rotation about the pseudo three-fold axis, which provided the best superimposing of residues c18–25 (Supplementary slide show). The c18–25 regions of yF1c10(II) and bF1c8 are well superimposed, which is consistent with AMP-PNP inhibited state of both enzymes. This region is involved in critical contacts with the C-terminal domain of the subunit bE, imposing the open conformation of this catalytic site. The rotors of yF1c10(I) and yF1c10(II) are clearly tilted with the bottom of the c-ring shifted by about 20 Å (Fig. 4A and B). Conversely, the yF1c10(II) and bF1c8 rotors better overlap. Regarding the c-rings as cylinders, their axes are parallel at 3.5 Å apart and the outer lateral surfaces are internally tangent (Fig. 4C and D). Obviously, the partial overlapping of the rings results from the larger radius of the c10-ring (28.5 Å) as compared with the c8-ring (25 Å). The common overlapping surface could be the trace of the actual interface between the c-ring and the subunit a. This permits to locate in F1-c-ring models, the subunit a and the peripheral stalk, which are lost during the yF1c10(II) crystallization process and the bF1c8 purification step. 3.3. The rotation axis of the rotor The c-ring can be considered as a rigid body fly wheel while the compliance of the F1-rotor is required for efficient energy transmission between FO and F1 (Neukirch et al., 2008). Moreover, on top of the a3b3 assembly, the end of the C-terminal helix of the subunit c

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Fig.3. Twist of the c subunit in yeast and bovine F1cn-ATPases. Stereo view of the C-terminal domains of the (ab)3 hexamer and the coiled coil of the c subunit following superimposition of the six N-terminal domains of yF1c10(I) (Pdb id: 2wpd, Dautant et al., 2010), yF1c10(II) (Pdb id: 3zry) and bF1c8 (Pdb id: 2xnd, Watt et al., 2010) structures colored in green, blue and red, respectively. The aTP and bDP subunits show the largest shifts and conformational changes (Table 2) and the c subunit rotation is also apparent. The view is from the c-ring.

Table 2 Rms differences in a-carbon positions in yF1c10(I) (Pdb id: 2wpd, Dautant et al., 2010) and bF1c8 (Pdb id: 2xnd, Watt et al., 2010) versus yF1c10(II) (Pdb id: 3zry) of structural domains⁄ following superimposition of the six N-terminal domains (a27–92, b10–25 and b30–82 for yeast; a25–90, b10–25 and b29–81 for bovine). The values in parentheses are the rmsd when individual domains are superimposed. The rmsd were calculated using the LSQMAN program (Kleywegt et al., 2001). Domains* yF1c10(I) versus yF1c10(II) N-terminal Nucleotide binding C-terminal bF1c8 versus yF1c10(II) N-terminal Nucleotide binding C-terminal

aTP

aDP

aE

bTP

bDP

bE

0.87 (0.49) 1.23 (0.49) 4.36 (0.86)

0.69 (0.42) 0.89 (0.38) 0.81 (0.47)

0.62 (0.34) 1.38 (0.37) 2.25 (0.48)

0.81 (0.42) 0.95 (0.40) 1.25 (0.41)

0.89 (0.42) 1.42 (0.41) 2.76 (0.44)

0.53 (0.38) 0.96 (0.46) 1.44 (0.50)

0.77 (0.48) 0.98 (0.57) 3.74 (1.93)

0.59 (0.43) 1.32 (0.66) 2.49 (1.20)

0.73 (0.40) 1.61 (0.64) 1.85 (1.10)

1.09 (0.89) 1.27 (0.53) 1.46 (0.72)

1.08 (0.85) 1.48 (0.55) 2.61 (0.73)

1.14 (0.89) 1.15 (0.52) 1.59 (1.00)

*

N-Terminal: yeast a27–92 and b10–82, bovine a25–90 and b10–81; Nucleotide binding domain: yeast a98–381 and b83–363, bovine a96–379 and b82–363; C-Terminal: yeast a382–509 and b364–474, bovine a380–507 and b364–474.

(c256–278) is constrained to lie inside a narrow bearing collar lined by conserved glycine/proline-rich loops. Though the c-ring has a mass of 77 kDa that is 55% of the rotor mass (128 kDa), its inertial moment (JDc-ring = 2.3 107 Å2 Da) is 66% of that of the rotor (JDrotor = 3.5  107 Å2 Da) as a consequence of the elongated shape of the central stalk along the rotor axis. The inertia center of the rotor is located within the c-ring, 5 Å below the F1–FO rotor interface. Therefore, it is more relevant to define the rotor axis using the cring axis. In yF1c10(I), the pseudo symmetry axis of the F1-stator is tilted by 11.4° from the rotor axis towards the subunit bDP and hence the c-ring axis runs at about 8 Å from the center of the bearing collar (Fig. 2A), which entails an unbalanced load. The shortest distance between the axes is 2.8 Å near the center of mass of the F1-central stalk. While in yF1c10(II) and bF1c8, the pseudo symmetry axes of the F1-stator are tilted 8° and 11° from the rotor axes, respectively, towards the subunit aTP and the c-ring axes pass through the center of the bearing collar, at 0.3 Å and 3 Å from the center of mass of the F1-stator of yF1c10(II) (Fig. 2B) and bF1c8, respectively (Supplementary slide show), which is a much more regular geometry for rotary engines. The overall shape of the sub-complex could result from the absence of the peripheral stalk in crystals. It has to be noted that in electron microscopy reconstructions of F1FO-ATP synthases, some of the class averages corresponding to certain views of F1FO show a tilt of F1 versus FO thus evidencing the flexibility of the assembly (Rubinstein et al., 2003; Lau et al., 2008; Böttcher et al., 2000).

To conclude, the F1 and the c-ring of yF1c10(II) and bF1c8 crystals appear appropriately coupled as expected for a functional rotary motor. 3.4. The F1–FO rotor interface On one side, an increasing number of high resolution structures of isolated membrane rotors of F-ATPases from various species (Preiss et al., 2010; Pogoryelov et al., 2010) are available with the c-ring stoichiometries varying from 8 to 15 and the top surface of the ring varying both in size and in shape. On the other side, structures of the yeast F1-ATPase showed the lower part of the subunit c more twisted than in the bovine enzyme. These two sets of structures provided split views of the two parts of the rotor. For the first time, with the bovine and the current yeast F1-c-ring structures, a valid comparison of F1–FO rotor interfaces can be done. In yF1c10(I), the inertia axes of the cde and c-ring parts are tilted 10° and not collinear (5 Å for the shortest distance) while in yF1c10(II) and bF1c8, they are less tilted, 8° and 5°, respectively, and almost collinear (less than 0.1 Å). At the bottom of the F1 sector, the foot of the stalk of yF1c10(II) is twisted along the rotor axis in the ATP hydrolysis direction by about 35° relative to bF1c8 (Fig. 4C and D). On the FO side, the yeast c-ring rolls by 10° in the same direction on the abutting surface relative to bovine, which could indicate a minor slide between FO-stators in intact complexes (Fig. 4C and D). The two rotations are combined to insure the coupling at the F1–FO rotor interface.

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Fig.4. Superimposition of yF1c10(I) and yF1c10(II) (parts A and B) and bovine bF1c8 and yF1c10(II) (parts C and D). yF1c10(I), yF1c10(II) and bF1c8 are shown in green, blue and red color, respectively. The pseudo three-fold axes of the F1-stators and the inertial axes of the c-rings are drawn and colored accordingly. The subunits a were used to superimpose the pseudo three-fold axes of yF1c10(II) with yF1c10(I) (Pdb id: 2wpd, Dautant et al., 2010) (left) and bF1c8 (Pdb id: 2xnd, Watt et al., 2010) (right). The top views are orthogonal to the mean plane of the two c-ring axes with vertical c10-ring rotor axis of yF1c10(II). At bottom, slabs of the F1 and FO rotor interfaces, are viewed from the intermembrane space. On parts C and D, arrows display the 35° torsion of the central stalk and the 10° roll of the c-ring of the yeast enzyme relative to the bovine ones (Watt et al., 2010). The c-rings of yF1c10(II) and bF1c8 are parallel and internally tangent and their axes are 3.5 Å apart. The putative location of the FO-stator was schematized at the periphery of the shared lateral surface (see text for explanation). (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.)

In any of the F1cn structures, three to four loop regions which link the two a-helices of each monomer c, are in tight contact with the subunits c and d while some of the subunits c on the opposite side of the ring show no clear contacts to the foot of the central stalk, leading to an asymmetry in the interface (Table S1). The buried surface area at the F1–FO rotor interface is 1136 Å2 and 1167 Å2 in bF1c8 and yF1c10(II), respectively. Whatever the stoichiometry, the F1–FO rotor interface areas of yeast and bovine are in the same range. The larger the F1–FO rotor interface is, the stronger the interaction between the two rotor part is. The smaller area (890 Å2) found in yF1c10(I) results from the slight opening between the two rotor parts by crystal packing forces.

3.5. Organization model of the ten predicted TMHs of the yeast membrane stator domain First, a hybrid model containing the yeast F1c10 and the bovine statorT was built using superimposed models of yF1c10(II) and bovine F1-statorT (Rees et al., 2009) and BDFsol (Kane Dickson et al., 2006) complexes, as described in Section 2. Then the current F1c10a-statorT model was docked into the cryo-EM map of intact F1FO-ATP synthase from yeast (Lau et al., 2008) displaying in the membrane region, next to ac10, an unoccupied volume suitable to put the missing TMHs of the FO stator. In a further step, using biochemical and topological data (Velours et al., 2000), a network organization of the missing TMHs can be deduced (Fig. 5). aTMH1 was added as an ideal a-helix in the periphery of the FO stator because the endogenous cysteine aC23 at the end of aTMH1 is able to

Fig.5. Slab view along the c-ring axis and from the intermembrane space, through the FO membranous domain, of the yF1c10(II) atomic model docked in the 24 Å resolution cryo-EM maps of the yeast F1FO-ATP synthase obtained in Dr. Rubinstein’s laboratory contoured at 5.5 r (Lau et al., 2008). The c-ring is drawn as red cartoon with cE59 sidechain in ball-and-stick. The large and small circles display the outer surfaces of the yeast and bovine c-ring, respectively. The model has been enhanced with TMHs of subunits a (5 TMHs), 4(b) (2 TMHs), i, f and 8 (1 TMH) drawn as cylinder and colored in blue, cyan, yellow, magenta and green, respectively. The subunit a is modeled according to the E. coli ac12-ring model (Pdb Id: 1c17, Rastogi and Girvin, 1999). The aR176 sidechain is drawn as ball-andstick. The FO stator subunits are based on known inter subunit disulfide crosslinking (see text). The scale bar represents 50 Å. (For interpretation of the references to colors in this figure legend, the reader is referred to the web version of this article.)

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form a disulfide bridge (aC23–aC23) in dimer preparations (Velours et al., 2011). Moreover, the observation of a dimeric subunit a associated with two c-rings (c10a2c10) in yeast led to the proposal that the subunit a may constitute an important part of the monomer–monomer interface (Wittig et al., 2008) appears as a peripheral subunit allowing its incorporation in a last step of assembly of the complex (Rak et al., 2007). Secondly, the subunit 4 with two TMHs (4TMH1–4TMH2) was added counterclockwise relative to the subunit a, viewed from F1, in agreement with mechanistic models of F-ATP synthase (Del Rizzo et al., 2006). The removal of 4TMH1 of the yeast subunit 4 does not alter the enzyme activity, which supports its location in the periphery of the stator (Soubannier et al., 2002). 4TMH2 extends out the inner mitochondria membrane as a long helix. In bovine enzyme, this long helix (residues 79–183) can bend around residue 146 to adopt a straight conformation (Kane Dickson et al., 2006; Rees et al., 2009). To put end to end 4TMH2 and the long helix, keeping the peripheral stalk within the cryo-EM map, the bend must be reduced even more. 4TMH2 comes out of the membrane about 10 Å from the edge of the c-ring. This is also supported by disulfide bridge formation between the 4D54C belonging to the 4TMH1/4TMH2 inter-helical loop) and the endogenous cysteine of subunit a in F1FO monomer (4D54C–aC23; Spannagel et al., 1998b) or the neighboring subunit 4 in dimer (4D54C–4D54C; Spannagel et al., 1998a). Finally, small subunits 8, f and i contain a single TMH (Roudeau et al., 1999; Velours et al., 2000). On one side of subunit 4, the subunit 8 is in proximity to subunits a, f and 4 in the intermembrane space (Stephens et al., 2003) and to subunit c. Subunit 8 is incorporated in an earlier step of complex assembly (Hadikusumo et al., 1988). On the other side, the non essential subunit i (Vaillier et al., 1999) interacts with subunit a (iK29C–aC23; Paumard et al., 2000) or the neighboring subunit i in dimer (iK51C–iK51C; Paumard et al., 2002) and thus could be located at the periphery of the enzyme. In the bovine F1-statorT complex, the truncated FO stator (statorT) has an elongated shape. It goes down along the non-catalytic aDPbTP interface and then winds counterclockwise, when viewed from the top of the F1 towards the membrane (Rees et al., 2009). In the current model, the N-terminal end of the subunit 4 at c-ring level underneath the subunit aDP of F1. When superimposing the pseudo three-fold axes of the (ab)3 domains of bF1c8 and yF1c10(II), the overlapping of the outer surfaces of the two c-ring suggests that it could be the location of the rotor/stator interface. The junction of the soluble and membrane parts of the peripheral stalk in the current hybrid F1FO model is in favor of the suggested interface location. Acknowledgments The authors would like to thank Dr. John Rubinstein (U. Toronto, Canada) for supplying the cryo-EM map of the yeast F1FO-ATP synthase. They wish also to thank the MX Group at SOLEIL (Saint-Aubin, France) and ESRF (Grenoble, France) for their help and support, particularly Matthew Bowler for dehydration experiments and helical data collection. Special thanks are due to JeanClaude Talbot for helpful discussions and insights. The project was supported by the Région Aquitaine, ESRF, SOLEIL and the Agence Nationale de la Recherche (ANR-06-PCVI-0016). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jsb.2011.10.015. References Abrahams, J.P., Leslie, A.G.W., Lutter, R., Walker, J.E., 1994. Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–668.

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