20 Aug 2010: Preiss L, Yildiz Ö, Hicks DB, Krulwich TA, Meier T (2010) Correction: A New Type of Proton Coordination in an F1Fo-ATP Synthase Rotor Ring. PLOS Biology 8(8): 10.1371/annotation/4b30bafe-631f-48dd-a5c2-4e727a5853d1. https://doi.org/10.1371/annotation/4b30bafe-631f-48dd-a5c2-4e727a5853d1 View correction
20 Aug 2010: Preiss L, Yildiz Ö, Hicks DB, Krulwich TA, Meier T (2010) Correction: A New Type of Proton Coordination in an F1Fo-ATP Synthase Rotor Ring. PLOS Biology 8(8): 10.1371/annotation/a025ce51-d0f4-41ca-b5b9-89d9c75cd26d. https://doi.org/10.1371/annotation/a025ce51-d0f4-41ca-b5b9-89d9c75cd26d View correction
We solved the crystal structure of a novel type of c-ring isolated from Bacillus pseudofirmus OF4 at 2.5 Å, revealing a cylinder with a tridecameric stoichiometry, a central pore, and an overall shape that is distinct from those reported thus far. Within the groove of two neighboring c-subunits, the conserved glutamate of the outer helix shares the proton with a bound water molecule which itself is coordinated by three other amino acids of outer helices. Although none of the inner helices contributes to ion binding and the glutamate has no other hydrogen bonding partner than the water oxygen, the site remains in a stable, ion-locked conformation that represents the functional state present at the c-ring/membrane interface during rotation. This structure reveals a new, third type of ion coordination in ATP synthases. It appears in the ion binding site of an alkaliphile in which it represents a finely tuned adaptation of the proton affinity during the reaction cycle.
Like the wind turbines that generate electricity, the F1Fo-ATP synthases are natural “ion turbines” each made up of a stator and a rotor that turns, when driven by a flow of ions, to generate the cell's energy supply of ATP. The Fo motor rotates by reversible binding and release of coupling ions that flow down the electrochemical ion gradient across the cytoplasmic cell membrane (in the case of bacteria) or intracellular organelle membranes (in the case of eukaryotic cells). Here, we present the structure of a rotor (c-)ring from a Bacillus species (B. pseudofirmus OF4) determined at high-resolution by X-ray crystallography. This bacterium prefers alkaline environments where the concentration of protons (H+) is lower outside than inside the cell – the inverse of the situation usually found in organisms that prefer neutral or acidic environments. The amino acid sequence of the protein subunits in this rotor, nevertheless, has features common to an important group of ATP synthases in organisms from bacteria to man. The structure reveals a new type of ion binding in which a protonated glutamate residue in the protein associates with a water molecule. This finding raises the possibility considered by Nobel laureate Paul Boyer several decades ago that a hydronium ion (a protonated water molecule, H3O+), rather than a proton alone, might be the coupling species that energizes ATP synthesis. Also, it demonstrates the finely tuned adaptation of ATP synthase rotor rings and their ion-binding sites to the specific requirements of different organisms.
Citation: Preiss L, Yildiz Ö, Hicks DB, Krulwich TA, Meier T (2010) A New Type of Proton Coordination in an F1Fo-ATP Synthase Rotor Ring. PLoS Biol 8(8): e1000443. https://doi.org/10.1371/journal.pbio.1000443
Academic Editor: John Kuriyan, University of California Berkeley, United States of America
Received: March 10, 2010; Accepted: June 24, 2010; Published: August 3, 2010
Copyright: © 2010 Preiss et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in parts by the Cluster of Excellence “Macromolecular Complexes” at the Goethe University Frankfurt (DFG Project EXC 115), the DFG Collaborative Research Center 807 (to TM), and a research grant GM28454 from the National Institute of General Medical Sciences (to TAK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ATP, adenosine 5'-triphosphate; DCCD, N,N'-dicyclohexyl-carbodiimide; NCD-4, N-cyclohexyl-N'-(4-dimethylamino-α-naphthyl)carbodiimide; pmf, proton motive force; smf, sodium motive force
Most living cells depend upon the adenosine triphosphate (ATP) generated by F1Fo-ATP synthases that are energized by a proton- or a sodium-motive force (pmf, smf). These multi-subunit enzymes contain a cytoplasmic F1 catalytic domain (subunits α3β3γδε) that is connected with a membrane-embedded Fo domain (ab2c10–15 in bacteria) by a central (γε) and peripheral (b2δ) stalk. Energetically down-hill ion translocation across the membrane through the Fo complex is mediated by successive interactions between the stator a-subunit and a rotor ring (c-ring). Translocation involves ion binding to an unoccupied c-subunit, rotation, and subsequent ion release. The c-ring is attached to the γε stalk subunits so that c-ring rotation causes rotation of the stalk. The inherently elastic  and asymmetric γ-subunit extends into the α3β3 headpiece  and by rotation  induces conformational changes  in the catalytic β-subunits, which results in ATP synthesis.
In the Na+-binding c11 ring from Ilyobacter tartaricus  and the H+-binding c15 ring from Spirulina platensis , the translocated ions are bound within the groove of two adjacent c-subunits in a coordination network including a conserved carboxylate (Glu). In both cases, the ion is further coordinated by a precise network of residues, several of which are common to both organisms (Figure 1). The ion specificity of these two systems is determined by several factors including the geometry and distances of the ion coordination network, and a water molecule  providing a coordination site for Na+. The ion binding specificity of ATP synthases in various cells is adapted to the physiological requirements of the organism, and the different binding motifs observed presumably reflect these adaptations. The range of the ion-binding motif includes variations from complete Na+-binding signatures to c-subunits where the conserved carboxylate (E/D) of the C-terminal helix is the only residue that can be predicted with confidence to play a role in ion coordination (e.g., in Escherichia coli or Homo sapiens, Figure 1). On this basis, we here assign the name “E/D-only” to the c-subunits of this sub-class of proton-coupled ATP synthases.
The c-subunits of selected species were aligned according to their cytoplasmic loop region (bold). Residues structurally proven to be involved in ion coordination shown in this work and in  are shown in dark red (Na+ or H+) and blue (H2O+H+). The conserved ion-binding glutamate/aspartate is highlighted in red for all species. The motifs (AxAxAxA and PxxExxP)  in Bacillus pseudofirmus OF4 (numbering) are highlighted in green. The type of ion binding in the respective ATP synthase is indicated on the right side, together with their corresponding binding motifs. Among these types: E/D-only is defined in the text, “Na+” is a sodium-coupled rotor with a complete Na+-binding motif , and “mix” is a proton-coupled rotor that has elements of the Na+ motif (highlighted in grey), e.g. a polar (or charged) residue at positions required for Na+ coordination ,.
Alkaliphilic Bacillus species are among the bacteria having proton-coupled ATP synthases  with E/D-only c-subunits (Figure 1). The extreme alkaliphile Bacillus pseudofirmus OF4 grows by oxidative phosphorylation with cytoplasmic pH values maintained 1.5–2.3 pH units below the high external pH (up to 11) of the medium . The existence of this reversed ΔpH poses a major thermodynamic problem, with which these cells must cope. Among a variety of adaptive strategies to resolve the energetic problem , some special adaptations of the ATP synthase itself have evolved: latent ATPase activity ,, a-subunit modification ,, and in particular, specific adaptations of the c-subunit sequence  resulting in a large c-ring width with more c-subunits . The adaptations to alkaliphilic conditions in an ATP synthase rotor, with a widely found but structurally uncharacterized E/D-only motif, made its c-ring an attractive candidate for an X-ray diffraction study.
Structure of the Bacillus pseudofirmus OF4 c13 Ring
Three-dimensional crystals of the c-ring from native Bacillus pseudofirmus OF4 F1Fo-ATP synthase were obtained and diffracted to 2.5 Å (Table 1). An asymmetric unit contains one complete c-ring, forming crystal contacts with three neighboring, laterally translated c-rings including two loop-to-loop contacts and one loop-to-C-terminus contact. One c-ring is formed by 13 identical c-subunits with a central pore (Figure 2A). Because of the unusually short N- and C-termini typical of Bacillus species (Figure 1), it does not extend into the periplasmic space but instead ends at the periplasmic membrane surface, whereas on the cytoplasmic side, the protrusion out of the membrane is comparable to that observed in other c-rings (Figure S1). In contrast with the distinctly hour-glass shape of the c11, K10, and c15 rotor rings ,,, this c13 ring resembles a “tulip beer glass” appearance, in which the only slightly curved periplasmic side transitions at about the depth of the ion-binding site into a more significantly flared cytoplasmic side (Figure 2B). The c13 ring has an outer diameter of 63 Å on the cytoplasmic side, 54 Å on the periplasmic side, and 52 Å in the middle. Each c-subunit consists of two α-helices that are connected by a short cytoplasmic loop (RQPE, residues 34 to 37). The N-terminal α-helices (residues 1 to 33) form an inner ring, surrounded by an outer ring of the C-terminal α-helices (residues 38 to 69) in staggered position. While the inner helices are remarkably straight (Figure 3A) and only slightly tilted by ∼5° toward the c-ring axis (as seen from the periplasm), the outer helices are more curved and have a kink in the middle of the helix at the key carboxylate residue (Glu54). Above that kink, toward the cytoplasmic loop, the outer helices are bent ∼30° to the membrane vertical and form a convex surface shape on the outside of the ring. Toward the periplasmic side the helices remain more straight but still tilt ∼10° against the membrane vertical (Figure 3). The outer surface of the c13 ring (Figure S2A) is encircled by a large hydrophobic region forming membrane borders at the level of Phe47 on the cytoplasmic side and Phe69 at the very end of the ring on the periplasmic side with a height of 34 Å. This region is the contact region of the c13 ring with the hydrophobic part of a membrane, in good agreement with previously described membrane borders for other c- (or K-) rings ,,. Except for the cytoplasmic end, the surface in the inner pore of the c13 ring is overall hydrophobic and binds detergent molecules (Figure S2B), which apparently replace phospholipids. A second leaflet of phospholipids/detergent molecules at the periplasmic side of the c-ring could only be discerned partially from the electron densities, but its existence can be inferred. In support of this notion are data showing a plug of phospholipids  in the I. tartaricus c11 ring and also atomic force microscopy topographs from Bacillus sp. strain TA2.A1 c13 ring .
The c-subunits are shown in different colors in ribbon representation. The protonated water molecules are shown as red spheres. (A) View perpendicular to the membrane from the cytoplasm. (B) Side view of the c13 ring. The membrane border is indicated with grey bars.
The c-subunits are shown in different colors as ribbons and the protonated water is shown as a red sphere. Coordination of the ion is indicated by dashed lines. (A) A c2-dimer consisting of two adjacent C-terminal α-helices (blue and yellow) and two straighter N-terminal helices (gray). (B) View perpendicular to the membrane focused on the binding side. (C) The binding site at pH 4.5, view from the membrane plane. The electron density (2Fobs-Fcalc, shown at 1.3 σ) is shown as a blue mesh. The coordinating residues Glu54, Leu52, Ala53, and Val56 are indicated. The omit map for the position of the water oxygen (O) is shown as purple mesh (σ = 4.5). Distances are in Å.
Special amino acid sequence motifs are found in the c-subunits of alkaliphilic Bacillus species (Figure 1). An altered glycine motif in the inner helices is shown to affect the structure and biochemistry of c-rings ,. The 63 Å diameter of the c13 ring width (Figure 2A) is larger than expected if compared with the diameters of the c11 and c15 c-rings ,, 50 Å and 65 Å, respectively, since it is closer to the diameter of the c15 ring. The more relaxed positioning of the helices in the alkaliphile ring is caused by the structural impact of the alanine motif AxAxAxA that replaces the glycine motif GxGxGxG most often found in the first helix coding region of c-subunits. Similarly, replacement of two glycines with serines also accounts for the larger than anticipated c-ring diameter observed in the ATP synthase of Bacillus TA2.A1 . On the outer helices of the c13 ring from an alkaliphile, two prolines located one helix turn below and above the ion-binding glutamate can be identified in a motif (PxxExxP)  in which the first proline, Pro51, is specific for alkaliphiles . Both prolines break the regular α-helix hydrogen bonding pattern and cause helix bends; these two motifs of this c-ring are important factors that have an impact on the c13 diameter and ion binding as well as the overall “tulip beer glass” shape of the complex (Figure S1).
The Ion Binding Site
Ion binding in all c-rings includes a conserved outer helix Glu (or Asp). In the c13 ring of B. pseudofirmus OF4, this residue (Glu54) is located ∼6 Å above the middle of the membrane (Figure 3) toward the cytoplasmic side. At the pH (4.5) used in the cryo-protection buffer, Glu54 is protonated. Two outer helices from neighboring c-subunits form an ion binding site (Figure 3C). During structure refinement a sphere-shaped density in 2Fobs-Fcalc as well as in the omit map remained unassigned. In close proximity of this density center (2.8-3.2 Å), four atoms were identified. One of these belongs to the side chain carboxyl oxygen (Oε2) of Glu54, whereas the three others originate from the backbone carbonyls of Leu52 and Ala53 and from the backbone nitrogen of Val56. The observed distances and the arrangement of the four associated hydrogen donor/acceptor sites around this density are in almost pyramidal arrangement and the hydrogen atom positions lie on a plane. Such an arrangement resembles internal protonated water molecules (hydronium ion)  in other proteins.
Electron densities of certain cations (e.g., Na+) or oxygen atoms from water molecules are similar and difficult to distinguish by X-ray crystallography at the given resolution. However, several lines of evidence indicate the density seen in the binding pocket of this c13 ring should be interpreted as water rather than Na+. The hydrogen-bonding distance and angle geometry for the ligands are in the typical range for water molecules in proteins, as they are also found, for example, in carboxypeptidase (Figure S3) or in the protonated water cluster of bacteriorhodopsin . In contrast, the mean distances for Na+ coordination such as those in the Na+-binding c11 ring  are significantly shorter (∼2.3 Å)  than observed in the c13 ring. For direct experimental evidence we used NCD-4, a fluorescent analogue of the ATP synthase inhibitor DCCD, which is known to react covalently with protonated glutamates/aspartates in c-subunits . Figure 4 illustrates the time-dependent labeling of detergent-solubilized c13 ring at pH 6.0. Consistent with dependence of labeling on protonation of the carboxylate, an increase of pH to 9.0 immediately and significantly decelerates the reaction. This observation has also been reported by others , and a control experiment showed that the labeling by NCD-4 continued to increase linearly when the labeling time was extended to 9,000 s without an alkaline pH shift (Figure S4). The marked deceleration upon imposition of an alkaline pH shift also resembles the DCCD labeling pattern of the proton-coupled c15 ring as a function of pH (unpublished data). Most importantly, the presence of 200 mM Na+ shows no dramatic influence on the labeling kinetics and addition of 200 mM K+ leads to comparable effects to Na+. Whereas the presence of salts, either Na+ or K+, presumably causes minor changes on, for example, detergent micelles, water availability, or fluorophore quenching , the lack of a major Na+-specific effect on NCD-4 binding rates contrasts sharply with the immediate and strong Na+-protective effect of a much lower Na+ concentration (15 mM) on the Na+-binding c11 ring . This key evidence for H+ rather than Na+ binding of the B. pseudofirmus OF4 c13 ring is fully consistent with biochemical studies on the pmf- (but not smf-) dependent B. pseudofirmus OF4 cells  and its H+-dependent ATP synthase . The result furthermore suggests that the ion binding site is highly selective for H+ over Na+ essentially under any (physiological) condition with a minimum concentration excess of ∼108 Na+ (200 mM) over H+ (pH 9.0). Taken together, the findings indicate that the density observed in the binding site of the c13 ring of the B. pseudofirmus OF4 ATP synthase is an oxygen atom with four valences. The data show that the proton must be located in between the atom positions of Glu54(Oε1/2) and the water oxygen (O).
The continuous fluorescence of a sample containing B. pseudofirmus c13 ring was recorded (yellow). The reaction of NCD-4 with Glu54 was started by the addition of 100 µM NCD-4 and the increase of fluorescence at λ = 465 nm was monitored. An increase of pH to 9.0 immediately and significantly decelerates the reaction. The presence of 200 mM NaCl (blue) had no notable effect beyond effects also made by the presence of 200 mM KCl (red) on the reaction kinetics.
Comparison of the c-Rings and Ion Binding Sites
In contrast to other rotor ring structures ,,, no residues from the inner helices contribute to ion coordination in the c13 ring (Figure 3B and Figure S1B). In the c11 and c15 rings a proline (Pro28 and Pro25, respectively) is involved in kinking the inner helices (not shown), thereby allowing the hydrogen bonding of the glutamine (Gln32 and Gln29, respectively) with the glutamate on the outer helix. This proline and glutamine are replaced by a glycine and valine, respectively, in the c13 ring. Consequently, the inner helices form a complete α-helical hydrogen bonding pattern, retain a straight shape, and cannot hydrogen bond with the ion-binding glutamate.
A second notable difference of the ion binding site in the c13 ring as compared with the c11 and c15 rings is visible at the second oxygen of the glutamate (Glu54 Oε1). Whereas in c11 and c15 this oxygen forms a hydrogen bond with a tyrosine from the adjacent outer helix, such an interaction is missing in the c13 ring (Figure 3C and Figure S1B). The hydrogen bonding network of Glu54 in the c13 ring is therefore reduced to one bond only compared to the others. The additional freedom allows more rotameric flexibility of the glutamate carboxylate. This property becomes impressively visible in an overlay of 13 single c-subunits taken from one asymmetric unit (Figure 5). Notably, although the carboxyl group appears in different rotameric states in the crystal structure, the distance of the closest Glu54 oxygen to the water oxygen remains in bonding distance (2.6–3.1 Å) across all binding sites of the c13 ring.
The color code is given in B-factor (temperature factor) from low (blue) to high (red). The zoomed region shows the ion binding site with the rotameric states of Glu54 as discussed in the text. The position of the water is indicated by an arrow. The position of the membrane border at the outer helix is indicated with grey bars.
The subtle but important differences in the H-bonding network geometry allow a fine-tuning of the pKa of the carboxylic acid  and serve to optimize the required solvation energy  which is necessary to unlock the site and allow ion release and reloading during the ion translocation mechanism in the Fo complex (Figure 6 and Text S1). Fine tuning of these parameters is of crucial importance within the a/c-ring interface, where the rotor binding sites pass a more hydrophilic environment  (and J. D. Faraldo-Gómez, personal communication with TM) that is somewhat unique because of the adaptations in both the a- and c-subunits of the alkaliphile ,,. By contrast, while the ion-binding site is in contact with the hydrophobic barrier of the lipid phase, during the long rotation cycle of the rotor ring, the glutamate is expected to be neutralized. The structural data suggest that under these conditions the ion binding site of the c13 ring remains in the ion-locked state, much in analogy with the Na+- and H+-locked states in the c11 and c15 ring, respectively ,.
The model shows the c13 ring (rotor, grey) with the neighboring a-subunit (stator, yellow) and the view is slanted to the membrane plane. A selection of c-subunits are shown with the ion coordinating Glu54 and helix 4 of the a-subunit  with Arg172 and Lys180 . In ATP synthesis mode of the enzyme, the rotor moves from left to right. Two access pathways to and from the binding sites in the membrane , are indicated in grey. The grey bars mark the membrane border. The four stages (0, 1, 2, and 3) of the ion translocation mechanism are indicated in a close-up view and further described in Text S1.
The amino acid residues involved in the coordination of the water are exactly at the same positions of the c13 ring as those that coordinate the water in the Na+-binding c11 ring (Figure 1 and Figure S1B) . This commonality of binding pattern underlines the evolutionary and functionally conserved relationship between the pmf- and smf-driven systems. The smf-driven ATP synthases have been suggested to be evolutionary pioneers in the establishment of the modern ATP synthases . If this hypothesis is correct, the E/D-only type such as that seen in the B. pseudofirmus OF4 c13 ring and in the mixed type (c15 ring) primarily found in light-driven systems (chloroplasts, cyanobacteria) could be derivatives of the c11 basic structure from an evolutionary point of view. Rather small differences in the amino acid sequences of the c-subunits apparently account for the different ion binding types, which are phenotypically manifested in the differently coupled and differently environmentally adapted ATP synthases.
A Hydronium Ion (H3O+) as Coupling Ion for F1Fo-ATP Synthases?
Hydrogen atoms have a very weak X-ray diffracting power and their electron density often does not match with the exact position of the nucleus. Therefore, from a crystallographic point of view, at current resolution, the data do not allow the distinction between a protonated glutamate associating with a water molecule and a hydronium ion as a separate species. The scenario of a hydronium ion as a possible coupling ion species in F1Fo-ATP synthases was proposed for consideration by Boyer more than 20 years ago . Later, experimental differences in the pH-dependent inhibition kinetics of Na+- and H+-ATP synthases were interpreted to be in support of this hypothesis , but recent high-resolution structure data on the cyanobacterial  and chloroplast  c-rings clearly conflict with this as a general hypothesis. The possibility raised by the structural data presented here that this E/D-only type of c-ring may ultimately conform to Boyer's suggestion awaits further experimental (and/or quantum mechanical) analyses.
This work shows a new type of proton coordination in an F1Fo-ATP synthase rotor ring. An additional electron density within the protonated ion binding site corresponds to a water molecule (but not Na+). It is evident that the coordination network of the water itself, in analogy with the stable water coordination network in the Na+-binding c11 ring, is a stabilizing and therefore a structural part of this c-ring. The presence of the water has been shown to enhance the Na+-binding affinity in the Na+-binding c11 ring . Given this observation we propose that the water in the c13 ring binding pocket also enhances the proton affinity. High affinity rotor binding sites are of central importance for all ATP synthases but are especially important for ATP synthases of bacteria that grow in alkaline environments ,.
Some of the novel details of this c-ring are likely to be specific to Bacillus species growing at high pH , especially those differences in shape that relate directly to alkaliphile-specific motifs. Perhaps the novel manner in which a water participates in proton binding is also a consequence of adaptation of the ATP synthase to alkaliphily. Further structural analysis of c-rings from the large groups of non-alkaliphilic species harboring the E/D-only motif is necessary to clarify the precise role of such water molecules in the ion translocation process. It may reveal the presence of this ion binding type throughout a broader subset of H+-coupled rotors, where it influences both ion affinity and selectivity during torque generation in the Fo motors of the H+-dependent F-type ATP synthases, and possibly also for the ion-driven motors known from V-type and A-type ATPases/synthases.
Materials and Methods
Purification and Crystallization of the c-Ring from Bacillus pseudofirmus OF4
The ATP synthase was purified from B. pseudofirmus OF4 in which a six histidine tag was inserted after the N-terminal methionine in the chromosomal gene encoding the β-subunit of the ATP synthase. The complex was extracted from everted vesicles with 1% β-dodecyl maltoside in the presence of 3 mg/ml soybean asolectin and purified by affinity chromatography on NiNTA agarose. The isolation of the c-ring was carried out according to . To improve purity of the sample, the c-ring was concentrated by ultrafiltration with an Amicon tube with a molecular weight cut-off of 10’000 (Millipore GmbH, Schwalbach, Germany), incubated with 1.5% Foscholine-12 (w/v) at 45°C for 10 min, and run on a sucrosegradient ,. The c-ring containing fractions were concentrated by Hydroxyapatite (BioGelHT, Bio-Rad, Munich, Germany)  and dialyzed for 12 h (10 mM Tris/HCl pH 8.0) at 4°C. The c-ring was further concentrated using polyethylene glycol (PEG)  to a final concentration of 2.5 mg/ml (bicinchoninic acid assay, Pierce, Rockford, IL, USA). Crystals were grown by vapor diffusion in hanging drops at 18°C to a size of approx. 200×100×100 µm3. The c-ring sample was supplied with 1% (w/v) of β-undecyl maltoside and mixed with crystallization buffer (0.1 M sodium acetate, pH 4.3) and 20% PEG 400 (v/v). Before flash-freezing in liquid nitrogen, the rod shaped clear crystals were transferred for 2 min into a buffer containing 30% PEG 400 (v/v), 0.1 M sodium acetate pH 4.5, and 0.05% β-dodecyl maltoside (w/v).
NCD-4 Labeling Reactions
A 60 µl sample (0.2 mg/ml) of purified c13 from Bacillus pseudofirmus OF4 in 12.5 mM MES-HCl (pH 6.0) buffer and 0.05% β-dodecyl maltoside (w/v) was used. Continuous increase of fluorescence was recorded with an F-4500 Hitachi Fluorescence Spectrophotometer (λex = 342 nm, λem = 465 nm). The reaction was started by the addition of 0.6 µl of NCD-4 (Invitrogen Inc.) from a 10 mM stock solution in dimethylformamide. After 2,000 s, the rate of reaction was greatly reduced by addition of 11 µl of 1 M Tris/HCl pH 9.0. The time required for the addition of these compounds (NCD-4 and Tris buffer) was approximately 5 s in both cases.
Data Collection, Structure Determination, and Refinement
Data to 2.5 Å resolution were collected from a single crystal at the Max-Planck beamline X10SA (PX-II) at the Swiss Light Source (SLS, Villigen, Schwitzerland) and processed using the XDS package (Kabsch, 1993). The structure was determined by molecular replacement using PHASER (McCoy, 2007) with two bundles of six subunits from the structure of the c15 ring from Spirulina platensis  as search model. Model bias was removed by density modification and solvent flattening with RESOLVE . Iterative cycles of model building and refinement were performed using COOT  and phenix.refine of the PHENIX package , respectively. During refinement, no non-crystallographic symmetry operation was applied. The refinement resulted in electron density maps that were unambiguously interpretable and after chain fitting the Ramachandran plot shows no outliers. Figures were generated using Povscript , POV-ray (http://www.povray.org), and Pymol . Electrostatic potential distribution was generated using Pymol .
Comparison of c-ring structures from Ilyobacter tartaricus, Bacillus pseudofirmus OF4, and Spirulina platensis. (A) The c-subunits are shown in ribbon representation. Side views of the c-rings from I. tartaricus (yellow, 1yce and 2wgm), B. pseudofirmus OF4 (blue, 2x2v), and S. platensis (green, 2wie). The membrane border is indicated with grey bars. (B) View on the three types of ion binding sites in F-type ATP synthases. c11, I. tartaricus; c13, B. pseudofirmus OF4; c15, S. platensis. The hydrogen bonding network is indicated by dashed lines and the ion/water molecules are shown with small spheres.
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Electrostatic potential distribution of the B. pseudofirmus OF4 c13 ring surface. (A) Side view on the surface. (B) Section through the ring, same view as in (A). Detergent molecules (Foscholine-12) attached to the hydrophobic inner surface are displayed in stick representation (yellow) and helices of the c-ring in ribbon representation. Colors: red, negative; blue, positive; white, neutral. The membrane border is indicated with grey bars.
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Comparison of ion coordination in B. pseudofirmus OF4 c13 ring and carboxypeptidase. (A) Ion coordination in the c13 ring from B. pseudofirmus OF4. (B) Ion coordination in carboxypeptidase A1 (PDB code 3i1u). The water oxygen at the glutamate is shown as a red sphere. Distances are given in Å. In both cases, the water oxygen has four valences for hydrogen, either four (A) or three (B) of them are forming a hydrogen bonding network with the corresponding protein (complex).
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Long-term kinetics of the modification of the ion-coordinating Glu54 of detergent-solubilized c13 ring from B. pseudofirmus OF4 with NCD-4 in the absence and presence of NaCl. The fluorescence of a sample containing B. pseudofirmus c13 ring was taken every 20 min at pH 6 in the absence (yellow circles) or presence (blue squares) of 200 mM NaCl. The reaction of NCD-4 with Glu54 was started by the addition of 100 µM NCD-4 and the increase of fluorescence at λ = 438 nm was followed for 9,000 s. The arrow marks the time point, at which the rate of NCD-4 labeling was reduced by shifting the pH to 9 in the experiment shown in Figure 4 (see text). For experimental details see Materials and Methods section.
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Werner Kühlbrandt is acknowledged for generous support of TM's research and helpful comments on the manuscript. The authors also thank José D. Faraldo-Gómez for sharing his unpublished simulation results and contributing to the development of the microscopic model of ring rotation. Denys Pogoryelov is acknowledged for providing models of the c15 ring used for molecular replacement trials. We furthermore would like to thank the staff of the Swiss Light Source (SLS, PXII) and the European Synchrotron Radiation Facility (ESRF) for their continuous support.
The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: LP DBH TAK TM. Performed the experiments: LP ÖY DBH. Analyzed the data: LP ÖY TM. Contributed reagents/materials/analysis tools: LP ÖY DBH. Directed the project and wrote the paper: TAK TM.
- 1. Junge W, Sielaff H, Engelbrecht S (2009) Torque generation and elastic power transmission in the rotary FOF1-ATPase. Nature 459: 364–370.W. JungeH. SielaffS. Engelbrecht2009Torque generation and elastic power transmission in the rotary FOF1-ATPase.Nature459364370
- 2. 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: 621–628.J. P. AbrahamsA. G. W. LeslieR. LutterJ. E. Walker1994Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria.Nature370621628
- 3. Noji H, Yasuda R, Yoshida M, Kinosita K Jr (1997) Direct observation of the rotation of F1-ATPase. Nature 386: 299–302.H. NojiR. YasudaM. YoshidaK. Kinosita Jr1997Direct observation of the rotation of F1-ATPase.Nature386299302
- 4. Boyer P. D (1993) The binding change mechanism for ATP synthase - some probabilities and possibilities. Biochim Biophys Acta 1140: 215–250.P. D. Boyer1993The binding change mechanism for ATP synthase - some probabilities and possibilities.Biochim Biophys Acta1140215250
- 5. Meier T, Polzer P, Diederichs K, Welte W, Dimroth P (2005) Structure of the rotor ring of F-Type Na+-ATPase from Ilyobacter tartaricus. Science 308: 659–662.T. MeierP. PolzerK. DiederichsW. WelteP. Dimroth2005Structure of the rotor ring of F-Type Na+-ATPase from Ilyobacter tartaricus.Science308659662
- 6. Pogoryelov D, Yildiz Ö, Faraldo-Gómez J. D, Meier T (2009) High-resolution structure of the rotor ring of a proton-dependent ATP synthase. Nat Struct Mol Biol 16: 1068–1073.D. PogoryelovÖ. YildizJ. D. Faraldo-GómezT. Meier2009High-resolution structure of the rotor ring of a proton-dependent ATP synthase.Nat Struct Mol Biol1610681073
- 7. Meier T, Krah A, Bond P. J, Pogoryelov D, Diederichs K, et al. (2009) Complete ion-coordination structure in the rotor ring of Na+-dependent F-ATP synthases. J Mol Biol 391: 498–507.T. MeierA. KrahP. J. BondD. PogoryelovK. Diederichs2009Complete ion-coordination structure in the rotor ring of Na+-dependent F-ATP synthases.J Mol Biol391498507
- 8. Krulwich T. A, Hicks D. B, Swartz T, Ito M (2007) Bioenergetic adaptations that support alkaliphily. In: Gerday C, Glansdorff N, editors. Physiology and Biochemistry of Extremophiles. Washington DC: ASM Press. pp. 311–329.T. A. KrulwichD. B. HicksT. SwartzM. Ito2007Bioenergetic adaptations that support alkaliphilyC. GerdayN. GlansdorffPhysiology and Biochemistry of ExtremophilesWashington DCASM Press311329
- 9. Hicks D. B, Krulwich T. A (1990) Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+. J Biol Chem 265: 20547–20554.D. B. HicksT. A. Krulwich1990Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+.J Biol Chem2652054720554
- 10. Krulwich T. A, Ito M, Gilmour R, Hicks D. B, Guffanti A. A (1998) Energetics of alkaliphilic Bacillus species: physiology and molecules. Adv Microb Physiol 40: 401–438.T. A. KrulwichM. ItoR. GilmourD. B. HicksA. A. Guffanti1998Energetics of alkaliphilic Bacillus species: physiology and molecules.Adv Microb Physiol40401438
- 11. Cook G. M, Keis S, Morgan H. W, von Ballmoos C, Matthey U, et al. (2003) Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1. J Bacteriol 185: 4442–4449.G. M. CookS. KeisH. W. MorganC. von BallmoosU. Matthey2003Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.J Bacteriol18544424449
- 12. McMillan D. G. G, Keis S, Dimroth P, Cook G. M (2007) A specific adaptation in the a subunit of thermoalkaliphilic F1Fo-ATP synthase enables ATP synthesis at high pH but not at neutral pH values. J Biol Chem 282: 17395–17404.D. G. G. McMillanS. KeisP. DimrothG. M. Cook2007A specific adaptation in the a subunit of thermoalkaliphilic F1Fo-ATP synthase enables ATP synthesis at high pH but not at neutral pH values.J Biol Chem2821739517404
- 13. Wang Z, Hicks D. B, Guffanti A. A, Baldwin K, Krulwich T. A (2004) Replacement of amino acid sequence features of a- and c-subunits of ATP synthases of alkaliphilic Bacillus with the Bacillus consensus sequence results in defective oxidative phosphorylation and non-fermentative growth at pH 10.5. J Biol Chem 279: 26546–26554.Z. WangD. B. HicksA. A. GuffantiK. BaldwinT. A. Krulwich2004Replacement of amino acid sequence features of a- and c-subunits of ATP synthases of alkaliphilic Bacillus with the Bacillus consensus sequence results in defective oxidative phosphorylation and non-fermentative growth at pH 10.5.J Biol Chem2792654626554
- 14. Liu J, Fujisawa M, Hicks D. B, Krulwich T. A (2009) Characterization of the functionally critical AXAXAXA and PXXEXXP motifs of the ATP synthase c-subunit from an alkaliphilic Bacillus. J Biol Chem 284: 8714–8725.J. LiuM. FujisawaD. B. HicksT. A. Krulwich2009Characterization of the functionally critical AXAXAXA and PXXEXXP motifs of the ATP synthase c-subunit from an alkaliphilic Bacillus.J Biol Chem28487148725
- 15. Matthies D, Preiss L, Klyszejko A. L, Muller D. J, Cook G. M, et al. (2009) The c13 ring from a thermoalkaliphilic ATP synthase reveals an extended diameter due to a special structural region. J Mol Biol 388: 611–618.D. MatthiesL. PreissA. L. KlyszejkoD. J. MullerG. M. Cook2009The c13 ring from a thermoalkaliphilic ATP synthase reveals an extended diameter due to a special structural region.J Mol Biol388611618
- 16. Murata T, Yamato I, Kakinuma Y, Leslie A. G, Walker J. E (2005) Structure of the rotor of the V-Type Na+-ATPase from Enterococcus hirae. Science 308: 654–659.T. MurataI. YamatoY. KakinumaA. G. LeslieJ. E. Walker2005Structure of the rotor of the V-Type Na+-ATPase from Enterococcus hirae.Science308654659
- 17. Meier T, Matthey U, Henzen F, Dimroth P, Müller D. J (2001) The central plug in the reconstituted undecameric c cylinder of a bacterial ATP synthase consists of phospholipids. FEBS Lett 505: 353–356.T. MeierU. MattheyF. HenzenP. DimrothD. J. Müller2001The central plug in the reconstituted undecameric c cylinder of a bacterial ATP synthase consists of phospholipids.FEBS Lett505353356
- 18. Behr J. P, Dumas P, Moras D (1982) The H3O+ cation: molecular structure of an oxonium-macrocyclic polyether complex. J Am Chem Soc 104: 4540–4543.J. P. BehrP. DumasD. Moras1982The H3O+ cation: molecular structure of an oxonium-macrocyclic polyether complex.J Am Chem Soc10445404543
- 19. Garczarek F, Brown L. S, Lanyi J. K, Gerwert K (2005) Proton binding within a membrane protein by a protonated water cluster. Proc Natl Acad Sci U S A 102: 3633–3638.F. GarczarekL. S. BrownJ. K. LanyiK. Gerwert2005Proton binding within a membrane protein by a protonated water cluster.Proc Natl Acad Sci U S A10236333638
- 20. Harding M. M (2004) The architecture of metal coordination groups in proteins. Acta Crystallogr D Biol Crystallogr 60: 849–859.M. M. Harding2004The architecture of metal coordination groups in proteins.Acta Crystallogr D Biol Crystallogr60849859
- 21. Sebald W, Machleidt W, Wachter E (1980) N,N'-dicyclohexylcarbodiimide binds specifically to a single glutamyl residue of the proteolipid subunit of the mitochondrial adenosinetriphosphatases from Neurospora crassa and Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 77: 785–789.W. SebaldW. MachleidtE. Wachter1980N,N'-dicyclohexylcarbodiimide binds specifically to a single glutamyl residue of the proteolipid subunit of the mitochondrial adenosinetriphosphatases from Neurospora crassa and Saccharomyces cerevisiae.Proc Natl Acad Sci U S A77785789
- 22. Valiyaveetil F, Hermolin J, Fillingame R. H (2002) pH dependent inactivation of solubilized F1F0 ATP synthase by dicyclohexylcarbodiimide: pKa of detergent unmasked aspartyl-61 in Escherichia coli subunit c. Biochim Biophys Acta 1553: 296–301.F. ValiyaveetilJ. HermolinR. H. Fillingame2002pH dependent inactivation of solubilized F1F0 ATP synthase by dicyclohexylcarbodiimide: pKa of detergent unmasked aspartyl-61 in Escherichia coli subunit c.Biochim Biophys Acta1553296301
- 23. von Ballmoos C (2007) Alternative proton binding mode in ATP synthases. J Bioenerg Biomembr 39: 441–445.C. von Ballmoos2007Alternative proton binding mode in ATP synthases.J Bioenerg Biomembr39441445
- 24. Adenier A, Aaron J. J (2002) A spectroscopic study of the fluorescence quenching interactions between biomedically important salts and the fluorescent probe merocyanine 540. Spectrochim Acta A Mol Biomol Spectrosc 58: 543–551.A. AdenierJ. J. Aaron2002A spectroscopic study of the fluorescence quenching interactions between biomedically important salts and the fluorescent probe merocyanine 540.Spectrochim Acta A Mol Biomol Spectrosc58543551
- 25. Meier T, Matthey U, von Ballmoos C, Vonck J, Krug von Nidda T, et al. (2003) Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases. J Mol Biol 325: 389–397.T. MeierU. MattheyC. von BallmoosJ. VonckT. Krug von Nidda2003Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases.J Mol Biol325389397
- 26. Guffanti A. A, Krulwich T. A (1988) ATP synthesis is driven by an imposed delta pH or delta mu H+ but not by an imposed delta pNa+ or delta mu Na+ in alkalophilic Bacillus firmus OF4 at high pH. J Biol Chem 263: 14748–14752.A. A. GuffantiT. A. Krulwich1988ATP synthesis is driven by an imposed delta pH or delta mu H+ but not by an imposed delta pNa+ or delta mu Na+ in alkalophilic Bacillus firmus OF4 at high pH.J Biol Chem2631474814752
- 27. Scheiner S, Hillenbrand E. A (1985) Modification of pK values caused by change in H-bond geometry. Proc Natl Acad Sci U S A 82: 2741–2745.S. ScheinerE. A. Hillenbrand1985Modification of pK values caused by change in H-bond geometry.Proc Natl Acad Sci U S A8227412745
- 28. Warshel A, Aqvist J (1991) Electrostatic energy and macromolecular function. Annu Rev Biophys Biophys Chem 20: 267–298.A. WarshelJ. Aqvist1991Electrostatic energy and macromolecular function.Annu Rev Biophys Biophys Chem20267298
- 29. Steed P. R, Fillingame R. H (2009) Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase. J Biol Chem 284: 23243–23250.P. R. SteedR. H. Fillingame2009Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase.J Biol Chem2842324323250
- 30. Mulkidjanian A. Y, Galperin M. Y, Makarova K. S, Wolf Y. I, Koonin E. V (2008) Evolutionary primacy of sodium bioenergetics. Biol Direct 3: 13.A. Y. MulkidjanianM. Y. GalperinK. S. MakarovaY. I. WolfE. V. Koonin2008Evolutionary primacy of sodium bioenergetics.Biol Direct313
- 31. Boyer P. D (1988) Bioenergetic coupling to protonmotive force: should we be considering hydronium ion coordination and not group protonation? Trends Biochem Sci 13: 5–7.P. D. Boyer1988Bioenergetic coupling to protonmotive force: should we be considering hydronium ion coordination and not group protonation?Trends Biochem Sci1357
- 32. von Ballmoos C, Dimroth P (2007) Two distinct proton binding sites in the ATP synthase family. Biochemistry 46: 11800–11809.C. von BallmoosP. Dimroth2007Two distinct proton binding sites in the ATP synthase family.Biochemistry461180011809
- 33. Krah A, Pogoryelov D, Meier T, Faraldo-Gómez J. D (2010) On the structure of the proton-binding site in the Fo rotor of chloroplast ATP synthases. J Mol Biol 395: 20–27.A. KrahD. PogoryelovT. MeierJ. D. Faraldo-Gómez2010On the structure of the proton-binding site in the Fo rotor of chloroplast ATP synthases.J Mol Biol3952027
- 34. Hicks D. B, Liu J, Fujisawa M, Krulwich T. A (2010) F1Fo-ATP synthases of alkaliphilic bacteria: lessons from their adaptations. Biochim Biophys Acta. 1797. : 1362–1377.D. B. HicksJ. LiuM. FujisawaT. A. Krulwich2010F1Fo-ATP synthases of alkaliphilic bacteria: lessons from their adaptations.Biochim Biophys Acta179713621377
- 35. Meier T, Morgner N, Matthies D, Pogoryelov D, Keis S, et al. (2007) A tridecameric c ring of the ATP synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential. Mol Microbiol 65: 1181–1192.T. MeierN. MorgnerD. MatthiesD. PogoryelovS. Keis2007A tridecameric c ring of the ATP synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential.Mol Microbiol6511811192
- 36. Meier T, Ferguson S. A, Cook G. M, Dimroth P, Vonck J (2006) Structural investigations of the membrane-embedded rotor ring of the F-ATPase from Clostridium paradoxum. J Bacteriol 188: 7759–7764.T. MeierS. A. FergusonG. M. CookP. DimrothJ. Vonck2006Structural investigations of the membrane-embedded rotor ring of the F-ATPase from Clostridium paradoxum.J Bacteriol18877597764
- 37. Yildiz Ö, Vinothkumar K. R, Goswami P, Kühlbrandt W (2006) Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation. EMBO J 25: 3702–3713.Ö. YildizK. R. VinothkumarP. GoswamiW. Kühlbrandt2006Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation.EMBO J2537023713
- 38. Terwilliger T (2004) SOLVE and RESOLVE: automated structure solution, density modification and model building. J Synchrotron Radiat 11: 49–52.T. Terwilliger2004SOLVE and RESOLVE: automated structure solution, density modification and model building.J Synchrotron Radiat114952
- 39. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132.P. EmsleyK. Cowtan2004Coot: model-building tools for molecular graphics.Acta Crystallogr D Biol Crystallogr6021262132
- 40. Zwart P. H, Afonine P. V, Grosse-Kunstleve R. W, Hung L. W, Ioerger T. R, et al. (2008) Automated structure solution with the PHENIX suite. Methods Mol Biol 426: 419–435.P. H. ZwartP. V. AfonineR. W. Grosse-KunstleveL. W. HungT. R. Ioerger2008Automated structure solution with the PHENIX suite.Methods Mol Biol426419435
- 41. Fenn D, Ringe D, Petsko G. A (2003) POVScript+: a program for model and data visualization using persistence of vision ray-tracing. J Appl Crystallogr 46: 944–947.D. FennD. RingeG. A. Petsko2003POVScript+: a program for model and data visualization using persistence of vision ray-tracing.J Appl Crystallogr46944947
- 42. DeLano W. L (2002) W. L. DeLano2002The Pymol Molecular Graphics System (DeLano Scientific, San Carlos, CA). The Pymol Molecular Graphics System (DeLano Scientific, San Carlos, CA).
- 43. Valiyaveetil F. I, Fillingame R. H (1998) Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase. J Biol Chem 273: 16241–16247.F. I. ValiyaveetilR. H. Fillingame1998Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase.J Biol Chem2731624116247
- 44. Steed P. R, Fillingame R. H (2009) Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase. J Biol Chem 284: 23243–23250.P. R. SteedR. H. Fillingame2009Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase.J Biol Chem2842324323250
- 45. Lau W. C, Rubinstein J. L (2010) Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound VO motor. Proc Natl Acad Sci U S A 107: 1367–1372.W. C. LauJ. L. Rubinstein2010Structure of intact Thermus thermophilus V-ATPase by cryo-EM reveals organization of the membrane-bound VO motor.Proc Natl Acad Sci U S A10713671372