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Rotary ATPases: A New Twist to an Ancient Machine
TIBS 1193 No. of Pages 11
Special Issue: 40 Years of TiBS
Review
Rotary ATPases: A New Twist to an Ancient Machine Werner Kühlbrandt1,* and Karen M. Davies1 Rotary ATPases are energy-converting nanomachines found in the membranes of all living organisms. The mechanism by which proton translocation through the membrane drives ATP synthesis, or how ATP hydrolysis generates a transmembrane proton gradient, has been unresolved for decades because the structure of a critical subunit in the membrane was unknown. Electron cryomicroscopy (cryoEM) studies of two rotary ATPases have now revealed a hairpin of long, horizontal, membrane-intrinsic a-helices in the a-subunit next to the c-ring rotor. The horizontal helices create a pair of aqueous half-channels in the membrane that provide access to the proton-binding sites in the rotor ring. These recent findings help to explain the highly conserved mechanism of ion translocation by rotary ATPases. Rotary ATPases All living organisms depend on having an adequate supply of ATP, the ubiquitous energy-carrying compound, in their cells. ATP is produced by the F-type ATP synthase, an ancient nanometerscale rotary machine in the energy-converting membranes of cells and cell organelles. F-type ATP synthases of bacteria, mitochondria, and chloroplasts work by making use of a transmembrane electrochemical gradient to produce ATP from ADP and phosphate [1]. The eukaryotic vacuolar (Vtype) ATPases work in reverse, using the energy liberated by ATP hydrolysis to pump protons across a membrane to acidify cellular compartments [2]. The A-type ATPases of archaea and some extremophilic bacteria resemble the V-type structurally but their predominant function is in ATP synthesis, as for the F-type [3]. Most rotary ATPases utilize or create a proton gradient, but those of a few anaerobic bacteria [4] and archaea [3] use a sodium gradient instead. All types of rotary ATPase conform to essentially the same building plan (Figure 1). F-type ATPases comprise a roughly globular, water-soluble F1 head and the Fo subcomplex in the membrane. The F1 head contains three catalytic b- and three non-catalytic /-subunits that alternate in a hexameric (/b)3 ring. The Fo subcomplex consists of the c-ring rotor, the stator subunits a and b, and, in mitochondria, several other small, hydrophobic subunits. V-type and Atype ATPases correspondingly have a globular V1 (A1) catalytic head and a Vo (Ao) subcomplex with a rotor ring in the membrane. In all rotary ATPases, the central stalk links the catalytic head to the rotor ring. The rotor rings of F-type ATP synthases have eight to 15 c-subunits (Figure 2). Most c-subunits are hairpins of two hydrophobic transmembrane helices whereas the ring subunits of V- and A-type ATPases often have two or more transmembrane helix hairpins [5]. In addition to the central stalk, F-type ATPases have one peripheral stalk, which connects the catalytic head to the stator in the membrane to prevent idle rotation of the whole assembly. The A-type ATPases have two peripheral stalks and V-type ATPases three (Figure 1). Unlike the bacterial and chloroplast ATP synthases, or the V- and A-type ATPases, all known mitochondrial ATP synthases form dimers in the membrane [6]. The dimers are arranged in long
Trends in Biochemical Sciences, Month Year, Vol. xx, No. yy
Trends Rotary ATPases are ancient nanomachines that couple the translocation of protons across membranes to ATP synthesis or ATP hydrolysis. The mechanism of proton translocation through the membrane by rotary ATPases has been unresolved for decades because the structure of critical subunits was unknown. Recent electron cryomicroscopy (cryoEM) structures of rotary ATPases have revealed a pair of long, horizontal, membrane-intrinsic helices in subunit a next to the c-ring rotor. At the interface between the rotor and subunit a, the horizontal helices create two offset hydrophilic half-channels that provide access to the proton-binding sites in the rotor. On the basis of the new cryoEM structures, realistic models of how proton translocation drives rotation and ATP synthesis can now be formulated and tested.
1 Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany
*Correspondence: [email protected] (W. Kühlbrandt).
http://dx.doi.org/10.1016/j.tibs.2015.10.006 © 2015 Elsevier Ltd. All rights reserved.
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(A)
(B)
(C)
(D)
Peripheral stalk
Central stalk
F1
V1
A1
FO
VO
AO
Figure 1. Schematic Overview of Rotary ATPases. (A) Mitochondrial F-type dimer; (B) chloroplast or bacterial F-type monomer; (C) vacuolar (V-type) ATPase;[9_TD$IF] and (D) archaeal (A-type) ATPase. Red and orange, catalytic and non-catalytic band /- (or A and B) subunits; brown, central stalk; green, peripheral stalks; yellow, rotor ring; blue, subunit a. The two darker-blue circles denote the two horizontal a-subunit helices adjacent to the rotor ring.
rows along the tightly curved ridges of the mitochondrial inner membrane cristae. The dimer rows have a profound effect on respiratory activity, growth rates, and mitochondrial morphology [7,8]. The rows are instrumental for the formation of the inner membrane cristae, which increase the membrane area available for respiratory chain complexes. The cristal lumen can be considered a mitochondrial subcompartment that may assist ATP production through local variations in proton concentration [9,10].
High-Resolution X-Ray Structures Because of their central importance in biological energy conversion, F-type ATPases have been subject to intense scrutiny for decades. Mitchell's chemiosmotic hypothesis, which stipulates that the electrochemical gradient generated by the respiratory chain complexes provides the energy to drive ATP synthesis, was formulated in the 1960s [1]. In the 1980s, Boyer proposed the concept of ATP synthesis by rotary catalysis [11]. In the 1990s, the high-resolution structure of the bovine heart F1 ATP synthase by Leslie and Walker provided a firm structural basis for the rotary mechanism [12]. Their studies showed that the three /- and three b-subunits of the F1 Figure 2. Structures of ATPase Rotor Rings. Top row from left: dark blue, c8-
2
C8
C9
C10
C11
C12
C13
C14
C15
K10
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ring of bovine mitochondrial ATP synthase [20]; blue, c9-ring of Mycobacterium phlei [60]; light blue, c10-ring of Saccharomyces cerevisiae mitochondrial ATP synthase [51]. Middle row: turquoise, c11-ring of Ilyobacter tartaricus [15]; green, c12-ring of Bacillus pseudofirmus OF4 mutant [61]; lime green, c13-ring of B. pseudofirmus OF4 [62]. Bottom row: ochre, c14-ring of Pisum sativum chloroplast ATP synthase [63]; orange, c15-ring of Spirulina platense cyanobacterial ATP synthase [64]; red, K10-ring of Enterococcus hirae A-type ATPase [16]. The A-type K-subunit has two helix hairpins. One helix in each Ksubunit (pale pink) contains the ion-conducting glutamate. All other rings are of Ftype ATP synthases with one helix hairpin per c-subunit. [10_TD$IF]Filled black arrowheads indicate the 11–13-Å distance between adjacent ion-binding sites in the c-rings. In the larger K-ring of the A-type ATPase this distance is 21.5 Å (open arrowheads). All ring structures are viewed from the direction of the catalytic head. Bar, 10 Å.
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head alternate around the central stalk of the g-subunit. Single-molecule light microscopy of immobilized F1 heads with a fluorescent actin filament attached to the g subunit demonstrated elegantly that the central stalk does indeed rotate relative to the (/b)3 assembly on ATP hydrolysis, confirming Boyer's concept of rotary catalysis [13]. A few years later, the 4-Å X-ray structure of a partial yeast mitochondrial ATP synthase complex showed the F1 head[12_TD$IF] connected to the c10 rotor ring[13_TD$IF] by the central stalk [14]. Higher-resolution structures of two bacterial rotor rings, the c11-ring of a sodium-translocating F-type ATP synthase [15] and the related K10-ring of a V-type ATPase [16], enabled detailed analysis of where, and exactly how, the cations bind to the rotor. The cation-binding site in the c-subunit is defined by a conserved acidic side chain (Asp61 in Escherichia coli), which mutagenesis had shown to be essential for ATP synthesis [17]. In the rotor ring structures, the ion-binding sites are located well below the membrane surface. This means that the ions must reach their binding sites through aqueous channels in another subunit, such as subunit a, in the Fo subcomplex. More recently, X-ray structures have been determined for a peripheral stalk subcomplex from bovine heart ATP synthase [18], the bovine F1–peripheral stalk complex [19], the bovine F1–rotor ring complex [20], and the same complex from yeast at improved resolution [21]. Yet, the critical subunit a eluded crystallographic analysis because it dissociates from the assembly on extended exposure to detergent, which is a prerequisite for 3D crystallization. Consequently, important aspects of the mechanism by which proton translocation thorough the membrane drives c-ring rotation remained obscure. This situation changed only recently with the structure of a complete and active mitochondrial ATP synthase dimer from the chlorophyll-less green alga Polytomella, determined by single-particle cryoEM [22] (Figure 3A–D). This review discusses the new structure in the context of the[2_TD$IF] recently published cryoEM maps of V- and A-type ATPases[3_TD$IF] [14_TD$IF][30] against a rich background of what was previously known about F-type ATPases.
New CryoEM Structures CryoEM has undergone a revolution during the past 2 years [23]. Structures of macromolecular complexes can now be determined at high resolution, without crystals, from electron micrographs of single molecules in vitrified aqueous suspension. This development was brought about by a new generation of direct electron detectors that enable the recording of highresolution images of radiation-sensitive biological samples and improved, user-friendly imageprocessing software [24]. Structures up to 2.2-Å resolution have been reported for soluble complexes [25] and up to 3.4 Å for membrane proteins [26,27]. At a resolution close to 6.0 Å, the map of the Polytomella ATP synthase dimer reveals /-helices clearly as characteristic, elongated densities. In the Fo assembly, the 20 transmembrane helices of each c-ring rotor are well resolved, as are another six per protomer at the dimer interface. Between these two sets of transmembrane helices, a bundle of four long, horizontal, membraneintrinsic /-helices was discovered. The four horizontal helices run almost perpendicular to the transmembrane /-helices and are arranged in two hairpins. Earlier biochemical crosslinking studies had established that subunit a is next to the c-ring [28,29]. As there were no other protein densities within crosslinking distance of the c-rings, the two horizontal helices adjacent to the cring (Figure 3B) were assigned to the elusive subunit a [22]. A few months after the online publication of the Polytomella dimer structure, a 7–8-Å resolution cryoEM map of the yeast V-type ATPase in three different rotational states was reported [30] (Figure 3A,B). The membrane region of this map also revealed a pair of long, horizontal, membrane-embedded helices adjacent to the rotor ring (Figure 3B). A hairpin of long, horizontal helices in the membrane next to the rotor ring is therefore a conserved feature of the evolutionarily distant F- and V-type ATPases.
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F-type
V-type
A-type
(A)
(B)
(C)
(D)
(E) B E.c.
176 153 164 A.t. 242 M P.sp. 206 S.c. 153 T.t. 519 A P.f. 523 S.c. 754 V B.t. 701
------KELTLQPFNHWAFIPVNLILEGVSLLSKPVSLGLRLFGNMYAGELIFILIAGLLP-------------------------RYVEPAAFLLPINVLEDFTKPLSLSFRLFGNILADELVVGVLVA----------------------------KYIQPTPILLPINILEDFTKPLSLSFRLFGNILADELVVVVLVS----------------------FLLPAGVPLPLAPFLVLLELISYCFRALSLGIRLFANMMAGHSLVKILSGFAWTMLCMNDIFYFI --------HFIPGGTPWPMAFIFVPLETISYTFRAVSLGVRLWVNMLAGHTLLHILTGMALALPFSLGFFSMV --------LFVPAGTPLPLVPLLVIIETLSYFARAISLGLRLGSNILAGHLLMVILAGLTFNFMLIN-LFTLV WLQGLMYLGFGVFLLAVLMSRIWLMIPEIFTQAGHILSHIRIYAVGAAGGILAGLLTDVGFAIAERLGLVGVL VFGAGAVLFIIGEFKNNGLMALLLVISDFFGFVGNWLSYARLMALALATAGIALVINIMVQMIWGFKIGPVPINESVGGEQGPFNFGDVMIHQVIHTIEFCLNCISHTASYLRLWALSLAHAQLSSVLWDMTISNAFSSKNSGSP EDAEEPTEDEVFDFGDTMVHQAIHTIEYCLGCISNTASYLRLWALSLAHAQLSEVLWTMVIHIGLKVKSLAGG *: * : ::
230 197 208 307 271 217 541 594 827 774
B E.c.
--WWSQWILNVPWAIFHILIITLQAFIFMVLTIVYLSMASEEH--------------------------------LVPLIIPIPIMLLGVFTSAIQALVFATLAGAYIGEALADHH-------------------------------LVPLVVPIPVMFLGLFTSGIQALIFATLAAAYIGESMEGHH----------------------------GA-LGPLFIVLALTGLELGVAILQAYVFTILICIYLNDAINLH-----------------------------PATFGVCCLLSALVGLEYLVAVLQSGVFSILSTVYVGEFNHDKFIGPAAKIVKKIH----------------FG-FVPLAMILAIMMLEFAIGIIQGYVWAILTASYLKDAVYLH-----------------------------LGLLVAGVLHLLILLLTTLGHMLQPIRLLWVEFFTKFGFYEENGRPYRP---------------------------LGIIVGAIVFIGGHIFSTAINALGAFVHALRLHYVEFFGTFYSGEGRRFEPFAARREISELKIEKPGGE LAVMKVVFLFAMWFVLTVCILVFMEGTSAMLHALRLHWVEAMSKFFEGEGYAYEPFSFRAIIE------------LALFFIFAAFATLTVAILLIMEGLSAFLHALRLHWVEFQNKFYSGTGFKFLPFSFEHIREGKFD------
271 238 249 349 327 259 590 663 890 838
C.r.
C A.t.
231 198 209 A.t. 308 P.sp. 272 S.c. 218 T.t. 542 P.f. 595 S.c. 828 B.t. 775 C.r.
C A.t. M A V
Figure 3. Electron Cryomicroscopy (CryoEM) Maps of Three Different Rotary ATPases. (A) Side view of mitochondrial F-type ATP synthase dimer from Polytomella sp. at 6.2-Å resolution [22], EMDB 2852; Saccharomyces cerevisiae V-type ATPase at 7-Å resolution [30], EMDB 6285; and Thermus thermophilus A-type ATPase at 10-Å resolution [37], EMDB 5335. Yellow, rotor rings; blue, horizontal helices. (B) c-ring rotors (yellow) and horizontal helices (blue) in the membrane subcomplexes of the rotary ATPases shown in (A). (C) Matrix view of long, membrane-intrinsic helix densities in the subunit a/c-ring assembly of the Polytomella ATP synthase [22] with fitted helix models. Prolines at proposed helix ends
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Subunit a Topography The long horizontal helices in subunit a came as a surprise. It had been expected that these helices would run more or less perpendicular to the membrane, as do virtually all other /-helices in any other membrane protein. Hydropathy plots and sequence variation analysis had predicted four to seven transmembrane helices in the a-subunit [[15_TD$IF]32]. Sulfhydryl labeling of surfaceaccessible residues was consistent with five such helices [33,34]. Crosslinking of double cysteine mutants suggested a model where four of the predicted helices formed a vertical bundle, of which two were in contact with the c-ring [28,29,35]. More than two decades of painstaking biochemical crosslinking and labeling experiments were interpreted in terms of various numbers of vertical transmembrane helices in subunit a. This interpretation was seemingly corroborated by two separate structural studies at intermediate resolution published in 2012. A 7-Å projection map of a bacterial subunit a/c11-ring subcomplex was obtained by electron crystallography [36]. In such projection maps, only vertical transmembrane /-helices show up clearly [22] and thus the horizontal helices remained undetected. The other intermediate-resolution structure was a 10-Å cryoEM map of an intact A-type ATPase from Thermus thermophilus [37] (Figure 3A). In this lower-resolution map, a density region next to the L-ring rotor (equivalent to the F-type c-ring rotor) was interpreted as eight short vertical or slightly tilted helices. In a recent re-evaluation of the T. thermophilus map [30], two long membraneintrinsic helices were identified adjacent to the c-ring (Figure 3B). Therefore, the two long membrane-intrinsic helices next to the rotor are an evolutionarily preserved and apparently fundamental feature of all rotary ATPases.
Key Subunit a Residues Site-directed mutagenesis of the E. coli ATP synthase revealed several essential polar residues in subunit a (Figure 3E). The most critical residue was the strictly conserved arginine 210. Any change in this residue abolished proton-coupled ATP synthesis [38,39]. Mutation to a smaller or negatively charged residue resulted in unproductive dissipation of the proton gradient. Interestingly, the R210Q mutant was partially rescued if a conserved glutamine (Q252 in E. coli) was changed to arginine. Therefore, these two residues are likely to be next to each other in the structure [40]. Second site suppressor mutations highlighted a number of other functionally important residues in subunit a, including E219 and H245 in E. coli, which are conserved in bacterial and mitochondrial F-type ATPases [41] (Figure 3C–E). A fit of the C-terminal subunit a sequence to the long helix hairpin in the 6.2-Å Polytomella dimer map placed the strictly conserved arginine 239 (R210 in E. coli) next to the proton-accepting residue (D61 in E. coli) in the c-subunit (Figure 3C) [22]. Several prolines, which often break or initiate /-helices, were positioned at either end of the two longest hairpin helices (Figure 3C). The sequence can be fitted to the hairpin in two alternative ways. The proposed fit [22] placed the strictly conserved arginine in the helix on the matrix side and the conserved glutamine (Q294 in Polytomella, Q252 in E. coli) in the lumenal helix, with the C terminus at the end of the lumenal helix (Figure 3C). In the alternative fit [42], the C terminus would be at the end of the other long are dark red. (D) Side view of (C) with proposed locations of functionally important residues indicated in color. Red, c-ring glutamates; blue, strictly conserved subunit a arginine and exchangeable glutamine; green, conserved histidine and glutamate. In one proposed fit [22], the strictly conserved arginine is in the helix above. In the equally plausible alternative fit [42], it would be in the helix below. (E) Sequence alignment of a-subunit homologs from F-, V-, and A-type ATPases. Conserved residues are highlighted in dark blue or green. The sequence fitted to the long helix hairpin in the Polytomella asubunit [22] in (C) is highlighted in light blue. Red arrowhead, strictly conserved arginine; green arrowheads, crosslinked residues in the Escherichia coli sequence; orange, solvent-accessible residues; red, prolines.[1_TD$IF] Abbreviations: E.c., E. coli; C. r., Chlamydomonas rheinhardii; A.t., Arabidopsis thaliana; P. sp., Polytomella sp.; S.c., S. cerevisiae; T.t., T. thermophilus; P.f., Pyrococcus furiosus; B.t., Bos taurus; B, bacterial; C, chloroplast; M, mitochondrial; V, vacuolar; A, A-type ATPase. Bars: in A and B, 20 Å; in C and D, 10 Å.
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helix on the matrix side. The conserved arginine and glutamine would swap positions but remain in the same region next to the c-ring, as would the functionally important histidine or glutamate residue (H248 in Polytomella, E219 in E. coli) that is located 2.5 helix turns toward the hairpin loop (Figure 3D). A higher-resolution structure is required to differentiate unambiguously between these two alternatives.
Two Aqueous Half-Channels As early as the 1990s, two offset aqueous half-channels in subunit a were already proposed on the basis of functionally important polar residues [43] and mechanistic considerations [44]. In line with this notion, cysteine mutagenesis revealed groups of polar residues in subunits a and c that were sensitive to inhibition by heavy-metal ions, which indicates that they are solvent accessible [[16_TD$IF]31,45–48]. The 6.2-Å map of the Polytomella ATP synthase dimer shows the detergent shell surrounding the Fo subcomplex very clearly. For the most part, this shell is of uniform thickness and envelops the vertical and horizontal /-helices in the membrane almost entirely. At the interface between the asubunit and the c-ring rotor, where the long horizontal helices approach the membrane surface on the matrix side, the detergent layer recedes to form a slanting, wedge-shaped aqueous channel (Figure 4A,B). The channel narrows to a slit between the c-ring and the horizontal a-subunit helices and ends at the proposed position of the conserved arginine. Most of the solvent-accessible residues in the a- and c-subunits [[17_TD$IF]31,34,48] would line this aqueous cavity. On the opposite, lumenal side of the membrane, another smaller aqueous cavity is visible in the detergent belt (Figure 4C). The proposed a-subunit fit [22] places the functionally important histidine and glutamate (H248 in Polytomella, E219 in E. coli) in this cavity and so the lumenal (A)
Side (D)
Side
(B)
(C)
Matrix
Lumen
(E)
(F)
Cytoplasm
Vacuole
Figure 4. Aqueous Half-Channels. (A–C) Polytomella mitochondrial ATP synthase dimer [22]. (D–F) Saccharomyces cerevisiae vacuolar ATPase [30]. (A,D) Side views of the membrane region; (B,E) matrix or cytoplasmic half-channel; (C,F) lumenal or vacuolar half-channel. White boxes and circles mark the positions of the aqueous half-channels. Purple, detergent shell; grey, stalk subunits; yellow; rotor ring; blue, horizontal a-subunit helices; green, solvent-accessible c-subunit region in Escherichia coli; orange and red helix regions in (B), solvent-accessible residues in the E. coli a-subunit. Bars, 10 Å.
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channel would act as the access pathway for protons to the rotor ring [[18_TD$IF]43,49]. It is worth noting that the aqueous channels in the detergent belt might have escaped detection in a highresolution X-ray structure. It is common practice in protein crystallography to use a procedure called solvent flattening to improve the phase information. Solvent flattening weakens or obliterates disordered non-protein density, such as detergent micelles, in an X-ray structure. Close examination also reveals an aqueous half-channel in the detergent shell of the Saccharomyces cerevisiae V-type ATPase map [30]. The roughly square-shaped, 20-Å cavity is visible on the cytoplasmic side (equivalent to the matrix side in mitochondria) between the cytoplasmic ends of the horizontal helices and the base of one of the peripheral stalks (Figure 4D,E). There may also be a narrow channel on the vacuolar side of the V-type map between subunit a and the rotor ring (Figure 4F). If confirmed by future studies, this would mean that the two aqueous halfchannels, like the pair of long, horizontal subunit a helices, are conserved and essential features of all rotary ATPases.
Mechanics of c-Ring Rotation High-resolution X-ray structures and molecular dynamics simulations [50,51] indicate that the acidic side chain in the proton-binding site of the c-subunit can adopt two different rotamer conformations depending on its chemical environment (Figure 5). In a hydrophilic environment, the carboxyl group predominately faces outward, enabling proton exchange with the bulk medium. In a hydrophobic environment, the carboxyl group points into the ring, adopting a locked conformation that traps the bound proton [[19_TD$IF]15,50–52]. As the c-ring rotates in the Fo assembly, the environment of the proton-binding site changes periodically from hydrophilic to hydrophobic and back again (Figure 6). When exposed to the hydrophilic environment of the lumenal proton-access channel, the acidic side chain is in the open conformation and accepts a proton from the cristal lumen. The protonated side chain now adopts the locked conformation, allowing the c-subunit to enter the hydrophobic environment of the lipid bilayer. The strictly conserved arginine in the long horizontal a-subunit helix acts as an electrostatic barrier, preventing backward rotation and proton leakage [53]. When, after completing an almost full circle, the c-subunit reaches the hydrophilic environment of the matrix half-channel, the protonated side chain changes back to the open conformation and the proton escapes to the highpH medium of the matrix. The electrochemical membrane potential drives the rotation of the c-ring as in a turbine. If the proton concentration is higher on the lumenal side (pH 7–7.4) than in the mitochondrial matrix (pH 8), the c-ring rotates anticlockwise as seen from the F1 head. This is the ATP synthesis mode of operation. F-type ATPases can also operate in reverse [54]. In the reverse mode, ATP (A)
(B) Aqueous environment
Matrix
Open Lipid environment
Lumen
Locked
Figure 5. Protonated Residues in the Rotor Ring c-Subunit. (A) Side view of the Saccharomyces cerevisiae c-ring structure [51] color coded for surface charge. Red, acidic; blue, basic. The back box outlines the acidic proton-binding site. Grey bars indicate the approximate position of the lipid bilayer. (B) Open and locked conformations of c-ring glutamates. When exposed to an aqueous environment, the c-ring glutamates adopt an open rotamer conformation. When exposed to the hydrophobic environment of the lipid bilayer, the glutamates change to an inward-facing, locked conformation. Residues shown are Glu59, Thr61, and Ala22. Adapted from [51].
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Matrix cavity
Rotaon
Membrane
Lumenal cavity
Figure 6. Proposed Mechanism of Proton Translocation and c-Ring Rotation. Protons (‘plus’ sign within a red circle) enter the Fo subcomplex via the conserved histidine (light blue) in the long, horizontal a-subunit helix (blue) that is exposed to the lumenal aqueous half-channel. Red arrows mark the proposed proton path. Protons bind to the open conformation of the deprotonated, negatively charged glutamate (orange) of the c-subunit (curved yellow cylinders). The glutamate side chain changes to a closed conformation that locks the proton and can enter the hydrophobic environment of the lipid bilayer. After an almost full revolution of the c-ring (grey arrow), the glutamate encounters the high pH 8 environment of the matrix half-channel, where it changes back to the open conformation and is deprotonated. The process of protonation in the lumenal half channel and deprotonation in the matrix half-channel causes the ring to rotate in an anticlockwise direction as seen from the matrix. Backward rotation is prevented by the positive charge of the strictly conserved arginine (light blue). The arginine is located 2.5 helix turns (13.5 Å) downstream of the conserved histidine/ glutamate (Figure 3E). This distance matches the spacing between adjacent protonation sites in the c-rings of any known subunit stoichiometry (Figure 2). The functionally important arginine and histidine residues in the long a-subunit helix can thus interact with two adjacent c-subunits in the ring simultaneously, as required for proton translocation, c-ring rotation, and ATP synthesis by rotary catalysis.
hydrolysis in the catalytic F1 head drives the acidic side chains that take up protons from the matrix channel (or its equivalent) backward through the membrane toward the conserved arginine. The positively charged arginine would strip the protons off and they would escape through the lumenal channel (or its equivalent), generating an electrochemical proton gradient. In the cell, V-type ATPases appear to work only in this ATP hydrolysis mode, pumping protons from the cytoplasm (equivalent to the matrix in mitochondrial[4_TD$IF]) into the lumen of the vacuole. V-type ATPases operate in synthesis mode only under artificial conditions with a steep proton gradient [55,56]. The reason for this asymmetry is unknown but it is most likely related either to the different rotor ring geometries (Figure 2), or to the exact constellation of polar and charged side chains in the aqueous half-channels, which would differ between the F- and V-type ATPases.
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Advantages of Horizontal Helices
Outstanding Questions
Do the long horizontal helices offer any advantage for proton translocation over vertical transmembrane helices? As noted earlier, the horizontal helices seem to be instrumental in forming the offset lumenal and matrix half-channels, but this could equally well be achieved by vertical helices. Numerous secondary active transporters have aqueous half-channels that are open on one or the other side of the membrane, without the need for long horizontal helices [57,58]. One apparent advantage of the horizontal a-subunit helices is that they [20_TD$IF]fit the concave shape of the rotor at its narrowest diameter (Figure 6). All c-ring rotors are more or less hourglass shaped and in this way the functionally important side chains in the long horizontal helices can approach the protonation site in the c-subunit more easily and closely than if the helices were oriented vertically. However, the most compelling answer to the intriguing question of why the a-subunit helices have to be horizontal may be found by considering the two conserved, functionally important residues Arg239 and His248 in subunit a of Polytomella (Arg210 and Glu219 in E. coli). These residues are 2.5 helix turns apart in all F-type ATPases and therefore the distance between their side chains is in the range 12–14 Å. This spacing correlates well with the distance between the protonation sites in adjacent c-subunits of the rotor ring. A comparison of c-ring structures indicates distances between the protonated carboxyl groups ranging from 13.5 Å for the smaller c8 and c10 rings to around 11.1–11.5 Å for the larger c14- or c15-ring (Figure 2). In a horizontal helix, the two conserved residues are able to interact with two adjacent c-subunits simultaneously at a fixed distance, irrespective of the ring diameter (Figure 6). This would not be the case if the a-subunit helices were oriented vertically.
What are the high-resolution structures of the membrane subcomplexes of the F-, V-, and A-type ATPases? What is the exact position and orientation of conserved polar residues in the subunit a/rotor ring assembly? What is the[2_TD$IF] atomic structure of the aqueous half-channels at the a-subunit/rotor interface[5_TD$IF]? Why are mitochondrial ATP synthases dimeric? Why do V-type ATPases work only in ATP hydrolysis mode under physiological conditions? Why do V-type rotor ring subunits have four helices with only one protonatable glutamate?
Interestingly, the V- and A-type ATPase sequences show only the strictly conserved arginine, whereas the histidine is either not present or in a different position (Figure 3E). This correlates with the different, two-hairpin structure of the K-subunit in the Enterococcus hirae rotor ring (Figure 2), where the spacing between subsequent protonation sites is roughly twice that in the c-rings. The larger distance would preclude a simultaneous interaction with two residues that are 12–14 Å apart in the horizontal helix. This mismatch may be related to the fact that, unlike the Ftype ATP synthases, the V-type ATPases normally operate in ATP hydrolysis mode to pump protons. In summary, the main advantage of the long horizontal helices seems to be that they can accommodate rotor rings of all sizes. In addition, the long horizontal helices that partly wrap around the c-ring may provide a stable, low-friction interface for the ring to rotate against. The orthogonal arrangement of the long a-subunit residues and the c-ring rotor, and the position of the strictly conserved arginine, have been preserved over an extremely long evolutionary timescale. Sequence comparison (Figure 3E) indicates that the horizontal subunit a-helices are essentially invariant in all proton-driven ATP synthases, including those of photosynthetic cyanobacteria, which are thought to have remained essentially unchanged for 3 billion years [59]. The proton gradient across the photosynthetic membrane of these early organisms must have driven a nanomachine very much like the modern ATP synthases, as there is no evidence of any other means of harnessing a transmembrane proton gradient for ATP synthesis in biology. The arrangement of helices and the positions of critical residues at the subunit a/c interface has evidently proved so successful that any change was selected against in the course of evolution.
Concluding Remarks Recent cryoEM structures of three different rotary ATPases indicate a previously undiscovered pair of long, horizontal helices of the stator a-subunit in the membrane. The long helices cross the c-ring rotor at an angle of roughly 908 in an arrangement that works equally well for c-rings of all sizes. This enables ATP synthases in energy-converting membranes to utilize either a steep pH gradient with larger rings, as in cyanobacteria and chloroplasts, or a shallow pH gradient with small rotor rings, as in mitochondria.
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Polar residues in the long horizontal helices create two laterally offset aqueous cavities on opposite sides of the membrane at the interface between the a-subunit and the c-ring rotor of Ftype ATPases. The cavities mediate proton access to and from the ion-binding sites in the c-ring. In the mitochondrial F-type ATP synthase, protons entering the binding site through the lumenal cavity protonate the negatively charged glutamate in the c-subunit. The protonated glutamate adopts a locked conformation as the ring rotates through the hydrophobic lipid phase of the membrane. In the aqueous matrix cavity, the proton is released and escapes to the high-pH matrix. In this way, the electrochemical proton gradient between the neutral cristal lumen and the slightly basic matrix drives c-ring rotation and ATP synthesis in the catalytic F1 head. V-type ATPases operate in reverse, using the energy liberated by ATP hydrolysis to pump protons across the vacuolar or endosomal membrane against a pH gradient. The long subunit a helices and the aqueous half-channels have most likely been conserved for billions of years. Some important questions remain concerning the exact structure and interaction of subunits in the membrane subcomplexes of rotary ATPases[21_TD$IF] (see Outstanding Questions). Higher-resolution structures that resolve amino acid side chains in the membrane of intact, functional ATPases are required to answer these questions and to explain the ancient mechanism of rotary ATPases in detail. Acknowledgments The authors thank Janet Vonck, Thomas Meier, and Matteo Allegretti for comments on the manuscript and the Max Planck Society and the DFG Cluster of Excellence Macromolecular Complexes for funding.
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29. Moore, K.J. et al. (2008) Structural interactions between transmembrane helices 4 and 5 of subunit a and the subunit c Ring of Escherichia coli ATP synthase. J. Biol. Chem. 283, 31726– 31735 30. Zhao, J. et al. (2015) Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241–245 31. Moore, K.J. et al. (2008) The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism. J. Biol. Chem. 283, 13044–13052 32. Vik, S.B. and Dao, N.N. (1992) Prediction of transmembrane topology of F0 proteins from Escherichia coli F1F0 ATP synthase using variational and hydrophobic moment analyses. Biochim. Biophys. Acta 1140, 199–207 33. Valiyaveetil, F.I. and Fillingame, R.H. (1998) Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase. J. Biol. Chem. 273, 16241–16247 34. Wada, T. et al. (1999) A novel labeling approach supports the fivetransmembrane model of subunit a of the Escherichia coli ATP synthase. J. Biol. Chem. 274, 17353–17357 35. Schwem, B.E. and Fillingame, R.H. (2006) Cross-linking between helices within subunit a of Escherichia coli ATP synthase defines the transmembrane packing of a four-helix bundle. J. Biol. Chem. 281, 37861–37867
46. Angevine, C.M. et al. (2003) Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane. Proc. Natl. Acad. Sci. U.S.A. 100, 13179–13183 47. Angevine, C.M. et al. (2007) Aqueous access pathways in ATP synthase subunit. Reactivity of cysteine substituted into transmembrane helices 1, 3, and 5. J. Biol. Chem. 282, 9001–9007 48. Steed, P.R. and Fillingame, R.H. (2014) Residues in the polar loop of subunit c in Escherichia coli ATP synthase function in gating proton transport to the cytoplasm. J. Biol. Chem. 289, 2127–2138 49. Vik, S.B. et al. (1998) Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel. J. Biol. Chem. 273, 16229–16234 50. Pogoryelov, D. et al. (2010) Microscopic rotary mechanism of ion translocation in the Fo complex of ATP synthases. Nat. Chem. Biol. 6, 891–899 51. Symersky, J. et al. (2012) Structure of the c10 ring of the yeast mitochondrial ATP synthase in the open conformation. Nat. Struct. Mol. Biol. 19, 485–491 52. Meier, T. et al. (2011) A paradigmatic molecular machine. In Molecular Machines in Biology, pp. 208–238, Cambridge University Press 53. Mitome, N. et al. (2010) Essential arginine residue of the Fo-a subunit in FoF1-ATP synthase has a role to prevent the proton shortcut without c-ring rotation in the Fo proton channel. Biochem. J. 430, 171–177
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39. Cain, B.D. and Simoni, R.D. (1989) Proton translocation by the F1F0ATPase of Escherichia coli Mutagenic analysis of the a subunit. J. Biol. Chem. 264, 3292–3300 40. Hatch, L.P. et al. (1995) The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity. J. Biol. Chem. 270, 29407–29412 41. Vik, S.B. et al. (2000) A model for the structure of subunit a of the Escherichia coli ATP synthase and its role in proton translocation. Biochim. Biophys. Acta 1458, 457–466 42. Zhou, A. et al. (2015) Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM. Elife 6,Published online October 2015. http://dx.doi.org/10.7554/eLife.10180 43. Vik, S.B. and Antonio, B.J. (1994) A mechanism of proton translocation by F1F0 ATP synthases suggested by double mutants of the a subunit. J. Biol. Chem. 269, 30364–30369 44. Junge, W. et al. (1997) ATP synthase: an electrochemical transducer with rotatory mechanics. Trends Biochem. Sci. 22, 420–423 45. Angevine, C.M. and Fillingame, R.H. (2003) Aqueous access channels in subunit a of rotary ATP synthase. J. Biol. Chem. 278, 6066–6074
57. Wöhlert, D. et al. (2014) Structure and substrate ion binding in the sodium/proton antiporter PaNhaP. Elife 3, e03579 58. Verdon, G. et al. (2014) Coupled ion binding and structural transitions along the transport cycle of glutamate transporters. Elife 3, e02283 59. Hohmann-Marriott, M.F. and Blankenship, R.E. (2011) Evolution of photosynthesis. Annu. Rev. Plant Biol. 62, 515–548 60. Preiss, L. et al. (2015) Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci. Adv. 1, e1500106 61. Preiss, L. et al. (2013) The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4. Proc. Natl. Acad. Sci. U.S.A. 110, 7874–7879 62. Preiss, L. et al. (2014) The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus OF4 is adapted to alkaliphilic lifestyle. Mol. Microbiol. 92, 973–984 63. Saroussi, S. et al. (2012) Structure and flexibility of the C-ring in the electromotor of rotary F0F1-ATPase of pea chloroplasts. PLoS One 7, e43045 64. Pogoryelov, D. et al. (2009) High-resolution structure of the rotor ring of a proton-dependent ATP synthase. Nat. Struct. Mol. Biol. 16, 1068–1073
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Special Issue: 40 Years of TiBS
Review
Rotary ATPases: A New Twist to an Ancient Machine Werner Kühlbrandt1,* and Karen M. Davies1 Rotary ATPases are energy-converting nanomachines found in the membranes of all living organisms. The mechanism by which proton translocation through the membrane drives ATP synthesis, or how ATP hydrolysis generates a transmembrane proton gradient, has been unresolved for decades because the structure of a critical subunit in the membrane was unknown. Electron cryomicroscopy (cryoEM) studies of two rotary ATPases have now revealed a hairpin of long, horizontal, membrane-intrinsic a-helices in the a-subunit next to the c-ring rotor. The horizontal helices create a pair of aqueous half-channels in the membrane that provide access to the proton-binding sites in the rotor ring. These recent findings help to explain the highly conserved mechanism of ion translocation by rotary ATPases. Rotary ATPases All living organisms depend on having an adequate supply of ATP, the ubiquitous energy-carrying compound, in their cells. ATP is produced by the F-type ATP synthase, an ancient nanometerscale rotary machine in the energy-converting membranes of cells and cell organelles. F-type ATP synthases of bacteria, mitochondria, and chloroplasts work by making use of a transmembrane electrochemical gradient to produce ATP from ADP and phosphate [1]. The eukaryotic vacuolar (Vtype) ATPases work in reverse, using the energy liberated by ATP hydrolysis to pump protons across a membrane to acidify cellular compartments [2]. The A-type ATPases of archaea and some extremophilic bacteria resemble the V-type structurally but their predominant function is in ATP synthesis, as for the F-type [3]. Most rotary ATPases utilize or create a proton gradient, but those of a few anaerobic bacteria [4] and archaea [3] use a sodium gradient instead. All types of rotary ATPase conform to essentially the same building plan (Figure 1). F-type ATPases comprise a roughly globular, water-soluble F1 head and the Fo subcomplex in the membrane. The F1 head contains three catalytic b- and three non-catalytic /-subunits that alternate in a hexameric (/b)3 ring. The Fo subcomplex consists of the c-ring rotor, the stator subunits a and b, and, in mitochondria, several other small, hydrophobic subunits. V-type and Atype ATPases correspondingly have a globular V1 (A1) catalytic head and a Vo (Ao) subcomplex with a rotor ring in the membrane. In all rotary ATPases, the central stalk links the catalytic head to the rotor ring. The rotor rings of F-type ATP synthases have eight to 15 c-subunits (Figure 2). Most c-subunits are hairpins of two hydrophobic transmembrane helices whereas the ring subunits of V- and A-type ATPases often have two or more transmembrane helix hairpins [5]. In addition to the central stalk, F-type ATPases have one peripheral stalk, which connects the catalytic head to the stator in the membrane to prevent idle rotation of the whole assembly. The A-type ATPases have two peripheral stalks and V-type ATPases three (Figure 1). Unlike the bacterial and chloroplast ATP synthases, or the V- and A-type ATPases, all known mitochondrial ATP synthases form dimers in the membrane [6]. The dimers are arranged in long
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Trends Rotary ATPases are ancient nanomachines that couple the translocation of protons across membranes to ATP synthesis or ATP hydrolysis. The mechanism of proton translocation through the membrane by rotary ATPases has been unresolved for decades because the structure of critical subunits was unknown. Recent electron cryomicroscopy (cryoEM) structures of rotary ATPases have revealed a pair of long, horizontal, membrane-intrinsic helices in subunit a next to the c-ring rotor. At the interface between the rotor and subunit a, the horizontal helices create two offset hydrophilic half-channels that provide access to the proton-binding sites in the rotor. On the basis of the new cryoEM structures, realistic models of how proton translocation drives rotation and ATP synthesis can now be formulated and tested.
1 Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Strasse 3, 60438 Frankfurt am Main, Germany
*Correspondence: [email protected] (W. Kühlbrandt).
http://dx.doi.org/10.1016/j.tibs.2015.10.006 © 2015 Elsevier Ltd. All rights reserved.
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(A)
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VO
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Figure 1. Schematic Overview of Rotary ATPases. (A) Mitochondrial F-type dimer; (B) chloroplast or bacterial F-type monomer; (C) vacuolar (V-type) ATPase;[9_TD$IF] and (D) archaeal (A-type) ATPase. Red and orange, catalytic and non-catalytic band /- (or A and B) subunits; brown, central stalk; green, peripheral stalks; yellow, rotor ring; blue, subunit a. The two darker-blue circles denote the two horizontal a-subunit helices adjacent to the rotor ring.
rows along the tightly curved ridges of the mitochondrial inner membrane cristae. The dimer rows have a profound effect on respiratory activity, growth rates, and mitochondrial morphology [7,8]. The rows are instrumental for the formation of the inner membrane cristae, which increase the membrane area available for respiratory chain complexes. The cristal lumen can be considered a mitochondrial subcompartment that may assist ATP production through local variations in proton concentration [9,10].
High-Resolution X-Ray Structures Because of their central importance in biological energy conversion, F-type ATPases have been subject to intense scrutiny for decades. Mitchell's chemiosmotic hypothesis, which stipulates that the electrochemical gradient generated by the respiratory chain complexes provides the energy to drive ATP synthesis, was formulated in the 1960s [1]. In the 1980s, Boyer proposed the concept of ATP synthesis by rotary catalysis [11]. In the 1990s, the high-resolution structure of the bovine heart F1 ATP synthase by Leslie and Walker provided a firm structural basis for the rotary mechanism [12]. Their studies showed that the three /- and three b-subunits of the F1 Figure 2. Structures of ATPase Rotor Rings. Top row from left: dark blue, c8-
2
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ring of bovine mitochondrial ATP synthase [20]; blue, c9-ring of Mycobacterium phlei [60]; light blue, c10-ring of Saccharomyces cerevisiae mitochondrial ATP synthase [51]. Middle row: turquoise, c11-ring of Ilyobacter tartaricus [15]; green, c12-ring of Bacillus pseudofirmus OF4 mutant [61]; lime green, c13-ring of B. pseudofirmus OF4 [62]. Bottom row: ochre, c14-ring of Pisum sativum chloroplast ATP synthase [63]; orange, c15-ring of Spirulina platense cyanobacterial ATP synthase [64]; red, K10-ring of Enterococcus hirae A-type ATPase [16]. The A-type K-subunit has two helix hairpins. One helix in each Ksubunit (pale pink) contains the ion-conducting glutamate. All other rings are of Ftype ATP synthases with one helix hairpin per c-subunit. [10_TD$IF]Filled black arrowheads indicate the 11–13-Å distance between adjacent ion-binding sites in the c-rings. In the larger K-ring of the A-type ATPase this distance is 21.5 Å (open arrowheads). All ring structures are viewed from the direction of the catalytic head. Bar, 10 Å.
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head alternate around the central stalk of the g-subunit. Single-molecule light microscopy of immobilized F1 heads with a fluorescent actin filament attached to the g subunit demonstrated elegantly that the central stalk does indeed rotate relative to the (/b)3 assembly on ATP hydrolysis, confirming Boyer's concept of rotary catalysis [13]. A few years later, the 4-Å X-ray structure of a partial yeast mitochondrial ATP synthase complex showed the F1 head[12_TD$IF] connected to the c10 rotor ring[13_TD$IF] by the central stalk [14]. Higher-resolution structures of two bacterial rotor rings, the c11-ring of a sodium-translocating F-type ATP synthase [15] and the related K10-ring of a V-type ATPase [16], enabled detailed analysis of where, and exactly how, the cations bind to the rotor. The cation-binding site in the c-subunit is defined by a conserved acidic side chain (Asp61 in Escherichia coli), which mutagenesis had shown to be essential for ATP synthesis [17]. In the rotor ring structures, the ion-binding sites are located well below the membrane surface. This means that the ions must reach their binding sites through aqueous channels in another subunit, such as subunit a, in the Fo subcomplex. More recently, X-ray structures have been determined for a peripheral stalk subcomplex from bovine heart ATP synthase [18], the bovine F1–peripheral stalk complex [19], the bovine F1–rotor ring complex [20], and the same complex from yeast at improved resolution [21]. Yet, the critical subunit a eluded crystallographic analysis because it dissociates from the assembly on extended exposure to detergent, which is a prerequisite for 3D crystallization. Consequently, important aspects of the mechanism by which proton translocation thorough the membrane drives c-ring rotation remained obscure. This situation changed only recently with the structure of a complete and active mitochondrial ATP synthase dimer from the chlorophyll-less green alga Polytomella, determined by single-particle cryoEM [22] (Figure 3A–D). This review discusses the new structure in the context of the[2_TD$IF] recently published cryoEM maps of V- and A-type ATPases[3_TD$IF] [14_TD$IF][30] against a rich background of what was previously known about F-type ATPases.
New CryoEM Structures CryoEM has undergone a revolution during the past 2 years [23]. Structures of macromolecular complexes can now be determined at high resolution, without crystals, from electron micrographs of single molecules in vitrified aqueous suspension. This development was brought about by a new generation of direct electron detectors that enable the recording of highresolution images of radiation-sensitive biological samples and improved, user-friendly imageprocessing software [24]. Structures up to 2.2-Å resolution have been reported for soluble complexes [25] and up to 3.4 Å for membrane proteins [26,27]. At a resolution close to 6.0 Å, the map of the Polytomella ATP synthase dimer reveals /-helices clearly as characteristic, elongated densities. In the Fo assembly, the 20 transmembrane helices of each c-ring rotor are well resolved, as are another six per protomer at the dimer interface. Between these two sets of transmembrane helices, a bundle of four long, horizontal, membraneintrinsic /-helices was discovered. The four horizontal helices run almost perpendicular to the transmembrane /-helices and are arranged in two hairpins. Earlier biochemical crosslinking studies had established that subunit a is next to the c-ring [28,29]. As there were no other protein densities within crosslinking distance of the c-rings, the two horizontal helices adjacent to the cring (Figure 3B) were assigned to the elusive subunit a [22]. A few months after the online publication of the Polytomella dimer structure, a 7–8-Å resolution cryoEM map of the yeast V-type ATPase in three different rotational states was reported [30] (Figure 3A,B). The membrane region of this map also revealed a pair of long, horizontal, membrane-embedded helices adjacent to the rotor ring (Figure 3B). A hairpin of long, horizontal helices in the membrane next to the rotor ring is therefore a conserved feature of the evolutionarily distant F- and V-type ATPases.
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F-type
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(E) B E.c.
176 153 164 A.t. 242 M P.sp. 206 S.c. 153 T.t. 519 A P.f. 523 S.c. 754 V B.t. 701
------KELTLQPFNHWAFIPVNLILEGVSLLSKPVSLGLRLFGNMYAGELIFILIAGLLP-------------------------RYVEPAAFLLPINVLEDFTKPLSLSFRLFGNILADELVVGVLVA----------------------------KYIQPTPILLPINILEDFTKPLSLSFRLFGNILADELVVVVLVS----------------------FLLPAGVPLPLAPFLVLLELISYCFRALSLGIRLFANMMAGHSLVKILSGFAWTMLCMNDIFYFI --------HFIPGGTPWPMAFIFVPLETISYTFRAVSLGVRLWVNMLAGHTLLHILTGMALALPFSLGFFSMV --------LFVPAGTPLPLVPLLVIIETLSYFARAISLGLRLGSNILAGHLLMVILAGLTFNFMLIN-LFTLV WLQGLMYLGFGVFLLAVLMSRIWLMIPEIFTQAGHILSHIRIYAVGAAGGILAGLLTDVGFAIAERLGLVGVL VFGAGAVLFIIGEFKNNGLMALLLVISDFFGFVGNWLSYARLMALALATAGIALVINIMVQMIWGFKIGPVPINESVGGEQGPFNFGDVMIHQVIHTIEFCLNCISHTASYLRLWALSLAHAQLSSVLWDMTISNAFSSKNSGSP EDAEEPTEDEVFDFGDTMVHQAIHTIEYCLGCISNTASYLRLWALSLAHAQLSEVLWTMVIHIGLKVKSLAGG *: * : ::
230 197 208 307 271 217 541 594 827 774
B E.c.
--WWSQWILNVPWAIFHILIITLQAFIFMVLTIVYLSMASEEH--------------------------------LVPLIIPIPIMLLGVFTSAIQALVFATLAGAYIGEALADHH-------------------------------LVPLVVPIPVMFLGLFTSGIQALIFATLAAAYIGESMEGHH----------------------------GA-LGPLFIVLALTGLELGVAILQAYVFTILICIYLNDAINLH-----------------------------PATFGVCCLLSALVGLEYLVAVLQSGVFSILSTVYVGEFNHDKFIGPAAKIVKKIH----------------FG-FVPLAMILAIMMLEFAIGIIQGYVWAILTASYLKDAVYLH-----------------------------LGLLVAGVLHLLILLLTTLGHMLQPIRLLWVEFFTKFGFYEENGRPYRP---------------------------LGIIVGAIVFIGGHIFSTAINALGAFVHALRLHYVEFFGTFYSGEGRRFEPFAARREISELKIEKPGGE LAVMKVVFLFAMWFVLTVCILVFMEGTSAMLHALRLHWVEAMSKFFEGEGYAYEPFSFRAIIE------------LALFFIFAAFATLTVAILLIMEGLSAFLHALRLHWVEFQNKFYSGTGFKFLPFSFEHIREGKFD------
271 238 249 349 327 259 590 663 890 838
C.r.
C A.t.
231 198 209 A.t. 308 P.sp. 272 S.c. 218 T.t. 542 P.f. 595 S.c. 828 B.t. 775 C.r.
C A.t. M A V
Figure 3. Electron Cryomicroscopy (CryoEM) Maps of Three Different Rotary ATPases. (A) Side view of mitochondrial F-type ATP synthase dimer from Polytomella sp. at 6.2-Å resolution [22], EMDB 2852; Saccharomyces cerevisiae V-type ATPase at 7-Å resolution [30], EMDB 6285; and Thermus thermophilus A-type ATPase at 10-Å resolution [37], EMDB 5335. Yellow, rotor rings; blue, horizontal helices. (B) c-ring rotors (yellow) and horizontal helices (blue) in the membrane subcomplexes of the rotary ATPases shown in (A). (C) Matrix view of long, membrane-intrinsic helix densities in the subunit a/c-ring assembly of the Polytomella ATP synthase [22] with fitted helix models. Prolines at proposed helix ends
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Subunit a Topography The long horizontal helices in subunit a came as a surprise. It had been expected that these helices would run more or less perpendicular to the membrane, as do virtually all other /-helices in any other membrane protein. Hydropathy plots and sequence variation analysis had predicted four to seven transmembrane helices in the a-subunit [[15_TD$IF]32]. Sulfhydryl labeling of surfaceaccessible residues was consistent with five such helices [33,34]. Crosslinking of double cysteine mutants suggested a model where four of the predicted helices formed a vertical bundle, of which two were in contact with the c-ring [28,29,35]. More than two decades of painstaking biochemical crosslinking and labeling experiments were interpreted in terms of various numbers of vertical transmembrane helices in subunit a. This interpretation was seemingly corroborated by two separate structural studies at intermediate resolution published in 2012. A 7-Å projection map of a bacterial subunit a/c11-ring subcomplex was obtained by electron crystallography [36]. In such projection maps, only vertical transmembrane /-helices show up clearly [22] and thus the horizontal helices remained undetected. The other intermediate-resolution structure was a 10-Å cryoEM map of an intact A-type ATPase from Thermus thermophilus [37] (Figure 3A). In this lower-resolution map, a density region next to the L-ring rotor (equivalent to the F-type c-ring rotor) was interpreted as eight short vertical or slightly tilted helices. In a recent re-evaluation of the T. thermophilus map [30], two long membraneintrinsic helices were identified adjacent to the c-ring (Figure 3B). Therefore, the two long membrane-intrinsic helices next to the rotor are an evolutionarily preserved and apparently fundamental feature of all rotary ATPases.
Key Subunit a Residues Site-directed mutagenesis of the E. coli ATP synthase revealed several essential polar residues in subunit a (Figure 3E). The most critical residue was the strictly conserved arginine 210. Any change in this residue abolished proton-coupled ATP synthesis [38,39]. Mutation to a smaller or negatively charged residue resulted in unproductive dissipation of the proton gradient. Interestingly, the R210Q mutant was partially rescued if a conserved glutamine (Q252 in E. coli) was changed to arginine. Therefore, these two residues are likely to be next to each other in the structure [40]. Second site suppressor mutations highlighted a number of other functionally important residues in subunit a, including E219 and H245 in E. coli, which are conserved in bacterial and mitochondrial F-type ATPases [41] (Figure 3C–E). A fit of the C-terminal subunit a sequence to the long helix hairpin in the 6.2-Å Polytomella dimer map placed the strictly conserved arginine 239 (R210 in E. coli) next to the proton-accepting residue (D61 in E. coli) in the c-subunit (Figure 3C) [22]. Several prolines, which often break or initiate /-helices, were positioned at either end of the two longest hairpin helices (Figure 3C). The sequence can be fitted to the hairpin in two alternative ways. The proposed fit [22] placed the strictly conserved arginine in the helix on the matrix side and the conserved glutamine (Q294 in Polytomella, Q252 in E. coli) in the lumenal helix, with the C terminus at the end of the lumenal helix (Figure 3C). In the alternative fit [42], the C terminus would be at the end of the other long are dark red. (D) Side view of (C) with proposed locations of functionally important residues indicated in color. Red, c-ring glutamates; blue, strictly conserved subunit a arginine and exchangeable glutamine; green, conserved histidine and glutamate. In one proposed fit [22], the strictly conserved arginine is in the helix above. In the equally plausible alternative fit [42], it would be in the helix below. (E) Sequence alignment of a-subunit homologs from F-, V-, and A-type ATPases. Conserved residues are highlighted in dark blue or green. The sequence fitted to the long helix hairpin in the Polytomella asubunit [22] in (C) is highlighted in light blue. Red arrowhead, strictly conserved arginine; green arrowheads, crosslinked residues in the Escherichia coli sequence; orange, solvent-accessible residues; red, prolines.[1_TD$IF] Abbreviations: E.c., E. coli; C. r., Chlamydomonas rheinhardii; A.t., Arabidopsis thaliana; P. sp., Polytomella sp.; S.c., S. cerevisiae; T.t., T. thermophilus; P.f., Pyrococcus furiosus; B.t., Bos taurus; B, bacterial; C, chloroplast; M, mitochondrial; V, vacuolar; A, A-type ATPase. Bars: in A and B, 20 Å; in C and D, 10 Å.
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helix on the matrix side. The conserved arginine and glutamine would swap positions but remain in the same region next to the c-ring, as would the functionally important histidine or glutamate residue (H248 in Polytomella, E219 in E. coli) that is located 2.5 helix turns toward the hairpin loop (Figure 3D). A higher-resolution structure is required to differentiate unambiguously between these two alternatives.
Two Aqueous Half-Channels As early as the 1990s, two offset aqueous half-channels in subunit a were already proposed on the basis of functionally important polar residues [43] and mechanistic considerations [44]. In line with this notion, cysteine mutagenesis revealed groups of polar residues in subunits a and c that were sensitive to inhibition by heavy-metal ions, which indicates that they are solvent accessible [[16_TD$IF]31,45–48]. The 6.2-Å map of the Polytomella ATP synthase dimer shows the detergent shell surrounding the Fo subcomplex very clearly. For the most part, this shell is of uniform thickness and envelops the vertical and horizontal /-helices in the membrane almost entirely. At the interface between the asubunit and the c-ring rotor, where the long horizontal helices approach the membrane surface on the matrix side, the detergent layer recedes to form a slanting, wedge-shaped aqueous channel (Figure 4A,B). The channel narrows to a slit between the c-ring and the horizontal a-subunit helices and ends at the proposed position of the conserved arginine. Most of the solvent-accessible residues in the a- and c-subunits [[17_TD$IF]31,34,48] would line this aqueous cavity. On the opposite, lumenal side of the membrane, another smaller aqueous cavity is visible in the detergent belt (Figure 4C). The proposed a-subunit fit [22] places the functionally important histidine and glutamate (H248 in Polytomella, E219 in E. coli) in this cavity and so the lumenal (A)
Side (D)
Side
(B)
(C)
Matrix
Lumen
(E)
(F)
Cytoplasm
Vacuole
Figure 4. Aqueous Half-Channels. (A–C) Polytomella mitochondrial ATP synthase dimer [22]. (D–F) Saccharomyces cerevisiae vacuolar ATPase [30]. (A,D) Side views of the membrane region; (B,E) matrix or cytoplasmic half-channel; (C,F) lumenal or vacuolar half-channel. White boxes and circles mark the positions of the aqueous half-channels. Purple, detergent shell; grey, stalk subunits; yellow; rotor ring; blue, horizontal a-subunit helices; green, solvent-accessible c-subunit region in Escherichia coli; orange and red helix regions in (B), solvent-accessible residues in the E. coli a-subunit. Bars, 10 Å.
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channel would act as the access pathway for protons to the rotor ring [[18_TD$IF]43,49]. It is worth noting that the aqueous channels in the detergent belt might have escaped detection in a highresolution X-ray structure. It is common practice in protein crystallography to use a procedure called solvent flattening to improve the phase information. Solvent flattening weakens or obliterates disordered non-protein density, such as detergent micelles, in an X-ray structure. Close examination also reveals an aqueous half-channel in the detergent shell of the Saccharomyces cerevisiae V-type ATPase map [30]. The roughly square-shaped, 20-Å cavity is visible on the cytoplasmic side (equivalent to the matrix side in mitochondria) between the cytoplasmic ends of the horizontal helices and the base of one of the peripheral stalks (Figure 4D,E). There may also be a narrow channel on the vacuolar side of the V-type map between subunit a and the rotor ring (Figure 4F). If confirmed by future studies, this would mean that the two aqueous halfchannels, like the pair of long, horizontal subunit a helices, are conserved and essential features of all rotary ATPases.
Mechanics of c-Ring Rotation High-resolution X-ray structures and molecular dynamics simulations [50,51] indicate that the acidic side chain in the proton-binding site of the c-subunit can adopt two different rotamer conformations depending on its chemical environment (Figure 5). In a hydrophilic environment, the carboxyl group predominately faces outward, enabling proton exchange with the bulk medium. In a hydrophobic environment, the carboxyl group points into the ring, adopting a locked conformation that traps the bound proton [[19_TD$IF]15,50–52]. As the c-ring rotates in the Fo assembly, the environment of the proton-binding site changes periodically from hydrophilic to hydrophobic and back again (Figure 6). When exposed to the hydrophilic environment of the lumenal proton-access channel, the acidic side chain is in the open conformation and accepts a proton from the cristal lumen. The protonated side chain now adopts the locked conformation, allowing the c-subunit to enter the hydrophobic environment of the lipid bilayer. The strictly conserved arginine in the long horizontal a-subunit helix acts as an electrostatic barrier, preventing backward rotation and proton leakage [53]. When, after completing an almost full circle, the c-subunit reaches the hydrophilic environment of the matrix half-channel, the protonated side chain changes back to the open conformation and the proton escapes to the highpH medium of the matrix. The electrochemical membrane potential drives the rotation of the c-ring as in a turbine. If the proton concentration is higher on the lumenal side (pH 7–7.4) than in the mitochondrial matrix (pH 8), the c-ring rotates anticlockwise as seen from the F1 head. This is the ATP synthesis mode of operation. F-type ATPases can also operate in reverse [54]. In the reverse mode, ATP (A)
(B) Aqueous environment
Matrix
Open Lipid environment
Lumen
Locked
Figure 5. Protonated Residues in the Rotor Ring c-Subunit. (A) Side view of the Saccharomyces cerevisiae c-ring structure [51] color coded for surface charge. Red, acidic; blue, basic. The back box outlines the acidic proton-binding site. Grey bars indicate the approximate position of the lipid bilayer. (B) Open and locked conformations of c-ring glutamates. When exposed to an aqueous environment, the c-ring glutamates adopt an open rotamer conformation. When exposed to the hydrophobic environment of the lipid bilayer, the glutamates change to an inward-facing, locked conformation. Residues shown are Glu59, Thr61, and Ala22. Adapted from [51].
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Matrix cavity
Rotaon
Membrane
Lumenal cavity
Figure 6. Proposed Mechanism of Proton Translocation and c-Ring Rotation. Protons (‘plus’ sign within a red circle) enter the Fo subcomplex via the conserved histidine (light blue) in the long, horizontal a-subunit helix (blue) that is exposed to the lumenal aqueous half-channel. Red arrows mark the proposed proton path. Protons bind to the open conformation of the deprotonated, negatively charged glutamate (orange) of the c-subunit (curved yellow cylinders). The glutamate side chain changes to a closed conformation that locks the proton and can enter the hydrophobic environment of the lipid bilayer. After an almost full revolution of the c-ring (grey arrow), the glutamate encounters the high pH 8 environment of the matrix half-channel, where it changes back to the open conformation and is deprotonated. The process of protonation in the lumenal half channel and deprotonation in the matrix half-channel causes the ring to rotate in an anticlockwise direction as seen from the matrix. Backward rotation is prevented by the positive charge of the strictly conserved arginine (light blue). The arginine is located 2.5 helix turns (13.5 Å) downstream of the conserved histidine/ glutamate (Figure 3E). This distance matches the spacing between adjacent protonation sites in the c-rings of any known subunit stoichiometry (Figure 2). The functionally important arginine and histidine residues in the long a-subunit helix can thus interact with two adjacent c-subunits in the ring simultaneously, as required for proton translocation, c-ring rotation, and ATP synthesis by rotary catalysis.
hydrolysis in the catalytic F1 head drives the acidic side chains that take up protons from the matrix channel (or its equivalent) backward through the membrane toward the conserved arginine. The positively charged arginine would strip the protons off and they would escape through the lumenal channel (or its equivalent), generating an electrochemical proton gradient. In the cell, V-type ATPases appear to work only in this ATP hydrolysis mode, pumping protons from the cytoplasm (equivalent to the matrix in mitochondrial[4_TD$IF]) into the lumen of the vacuole. V-type ATPases operate in synthesis mode only under artificial conditions with a steep proton gradient [55,56]. The reason for this asymmetry is unknown but it is most likely related either to the different rotor ring geometries (Figure 2), or to the exact constellation of polar and charged side chains in the aqueous half-channels, which would differ between the F- and V-type ATPases.
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Advantages of Horizontal Helices
Outstanding Questions
Do the long horizontal helices offer any advantage for proton translocation over vertical transmembrane helices? As noted earlier, the horizontal helices seem to be instrumental in forming the offset lumenal and matrix half-channels, but this could equally well be achieved by vertical helices. Numerous secondary active transporters have aqueous half-channels that are open on one or the other side of the membrane, without the need for long horizontal helices [57,58]. One apparent advantage of the horizontal a-subunit helices is that they [20_TD$IF]fit the concave shape of the rotor at its narrowest diameter (Figure 6). All c-ring rotors are more or less hourglass shaped and in this way the functionally important side chains in the long horizontal helices can approach the protonation site in the c-subunit more easily and closely than if the helices were oriented vertically. However, the most compelling answer to the intriguing question of why the a-subunit helices have to be horizontal may be found by considering the two conserved, functionally important residues Arg239 and His248 in subunit a of Polytomella (Arg210 and Glu219 in E. coli). These residues are 2.5 helix turns apart in all F-type ATPases and therefore the distance between their side chains is in the range 12–14 Å. This spacing correlates well with the distance between the protonation sites in adjacent c-subunits of the rotor ring. A comparison of c-ring structures indicates distances between the protonated carboxyl groups ranging from 13.5 Å for the smaller c8 and c10 rings to around 11.1–11.5 Å for the larger c14- or c15-ring (Figure 2). In a horizontal helix, the two conserved residues are able to interact with two adjacent c-subunits simultaneously at a fixed distance, irrespective of the ring diameter (Figure 6). This would not be the case if the a-subunit helices were oriented vertically.
What are the high-resolution structures of the membrane subcomplexes of the F-, V-, and A-type ATPases? What is the exact position and orientation of conserved polar residues in the subunit a/rotor ring assembly? What is the[2_TD$IF] atomic structure of the aqueous half-channels at the a-subunit/rotor interface[5_TD$IF]? Why are mitochondrial ATP synthases dimeric? Why do V-type ATPases work only in ATP hydrolysis mode under physiological conditions? Why do V-type rotor ring subunits have four helices with only one protonatable glutamate?
Interestingly, the V- and A-type ATPase sequences show only the strictly conserved arginine, whereas the histidine is either not present or in a different position (Figure 3E). This correlates with the different, two-hairpin structure of the K-subunit in the Enterococcus hirae rotor ring (Figure 2), where the spacing between subsequent protonation sites is roughly twice that in the c-rings. The larger distance would preclude a simultaneous interaction with two residues that are 12–14 Å apart in the horizontal helix. This mismatch may be related to the fact that, unlike the Ftype ATP synthases, the V-type ATPases normally operate in ATP hydrolysis mode to pump protons. In summary, the main advantage of the long horizontal helices seems to be that they can accommodate rotor rings of all sizes. In addition, the long horizontal helices that partly wrap around the c-ring may provide a stable, low-friction interface for the ring to rotate against. The orthogonal arrangement of the long a-subunit residues and the c-ring rotor, and the position of the strictly conserved arginine, have been preserved over an extremely long evolutionary timescale. Sequence comparison (Figure 3E) indicates that the horizontal subunit a-helices are essentially invariant in all proton-driven ATP synthases, including those of photosynthetic cyanobacteria, which are thought to have remained essentially unchanged for 3 billion years [59]. The proton gradient across the photosynthetic membrane of these early organisms must have driven a nanomachine very much like the modern ATP synthases, as there is no evidence of any other means of harnessing a transmembrane proton gradient for ATP synthesis in biology. The arrangement of helices and the positions of critical residues at the subunit a/c interface has evidently proved so successful that any change was selected against in the course of evolution.
Concluding Remarks Recent cryoEM structures of three different rotary ATPases indicate a previously undiscovered pair of long, horizontal helices of the stator a-subunit in the membrane. The long helices cross the c-ring rotor at an angle of roughly 908 in an arrangement that works equally well for c-rings of all sizes. This enables ATP synthases in energy-converting membranes to utilize either a steep pH gradient with larger rings, as in cyanobacteria and chloroplasts, or a shallow pH gradient with small rotor rings, as in mitochondria.
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Polar residues in the long horizontal helices create two laterally offset aqueous cavities on opposite sides of the membrane at the interface between the a-subunit and the c-ring rotor of Ftype ATPases. The cavities mediate proton access to and from the ion-binding sites in the c-ring. In the mitochondrial F-type ATP synthase, protons entering the binding site through the lumenal cavity protonate the negatively charged glutamate in the c-subunit. The protonated glutamate adopts a locked conformation as the ring rotates through the hydrophobic lipid phase of the membrane. In the aqueous matrix cavity, the proton is released and escapes to the high-pH matrix. In this way, the electrochemical proton gradient between the neutral cristal lumen and the slightly basic matrix drives c-ring rotation and ATP synthesis in the catalytic F1 head. V-type ATPases operate in reverse, using the energy liberated by ATP hydrolysis to pump protons across the vacuolar or endosomal membrane against a pH gradient. The long subunit a helices and the aqueous half-channels have most likely been conserved for billions of years. Some important questions remain concerning the exact structure and interaction of subunits in the membrane subcomplexes of rotary ATPases[21_TD$IF] (see Outstanding Questions). Higher-resolution structures that resolve amino acid side chains in the membrane of intact, functional ATPases are required to answer these questions and to explain the ancient mechanism of rotary ATPases in detail. Acknowledgments The authors thank Janet Vonck, Thomas Meier, and Matteo Allegretti for comments on the manuscript and the Max Planck Society and the DFG Cluster of Excellence Macromolecular Complexes for funding.
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