ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase

Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, 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 (1995) 29407-29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N'-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F(1)-F(o) interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.     

A novel labeling approach supports the five-transmembrane model of subunit a of the Escherichia coli ATP synthase.

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBN-permeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.     

Cross-linking between helices within subunit a of Escherichia coli ATP synthase defines the transmembrane packing of a four-helix bundle

Subunit a of F(1)F(0) ATP synthase is required in the H(+) transport driven rotation of the c-ring of F(0), the rotation of which is coupled to ATP synthesis in F(1). The three-dimensional structure of subunit a is unknown. In this study, Cys substitutions were introduced into two different transmembrane helices (TMHs) of subunit a, and the proximity of the thiol side chains was tested via attempted oxidative cross-linking to form the disulfide bond. Pairs of Cys substitutions were made in TMHs 2/3, 2/4, 2/5, 3/4, 3/5, and 4/5. Cu(+2)-catalyzed oxidation led to cross-link formation between Cys pairs L120C(TMH2) and S144C(TMH3), L120C(TMH2) and G218C(TMH4), L120C(TMH2) and H245C(TMH5), L120C(TMH2) and I246C(TMH5), N148C(TMH3) and E219C(TMH4), N148C(TMH3) and H245C(TMH5), and G218C(TMH4) and I248C(TMH5). Iodine, but not Cu(+2), was found to catalyze cross-link formation between D119C(TMH2) and G218C(TMH4). The results suggest that TMHs 2, 3, 4, and 5 form a four-helix bundle with one set of key functional residues in TMH4 (Ser-206, Arg-210, and Asn-214) located at the periphery facing subunit c. Other key residues in TMHs 2, 4, and 5, which were concluded previously to compose a possible aqueous access pathway from the periplasm, were found to locate to the inside of the four-helix bundle.     

Characterization of the first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase by surface labeling,cross-linking,and mutagenesis

The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunit b, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40-64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits.     

The beta subunit of the Escherichia coli ATP synthase exhibits a tight membrane binding property

We have developed a chromatographic procedure to analyze the association of the subunits of the Escherichia coli F1Fo-ATP synthase with the cytoplasmic membrane. Minicells containing [35S]-labeled ATP synthase subunits are treated with lysozyme, solubilized, and chromatographed on a Sepharose CL-2B column in buffer containing urea and taurodeoxycholate. ATP synthase subunits are resolved into membrane intrinsic and membrane extrinsic subunits. Interestingly, a significant amount (36%) of the F1 subunit beta fractionates with the membrane intrinsic Fo subunits. About half of this amount (19%) of beta is non-specifically bound to the membrane. Interaction of beta with the membrane is not mediated by the amino terminal portion of beta.     

Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli

Expression of subunit III of the ATP synthase from spinach chloroplasts in Escherichia coli has been achieved. Although the protein is inserted into the bacterial cytoplasmic membrane, formation of a functional Fo complex was not observed.     

Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase.

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Labeling of subunit b of the ATP synthase from Escherichia coli with a photoreactive phospholipid analogue

Purified ATP synthase (F1F0) from Escherichia coli K12 was labeled with the hydrophobic photoreactive label 1-palmitoyl 2-(2-azido-4-nitro)benzoyl sn-glycero-3-[3H]phosphocholine in reconstituted proteoliposomes. The F0-subunit b was predominantly labeled. A very low amount of label was detected on the other F0-subunits a and c. The label in subunit b could be traced back by proteolytic digestion to the NH2-terminal fragment 1 to 53 which contains the stretch of hydrophobic amino acid residues 1 to 32. By sequencing the intact protein, the distribution of label among the amino acids in this segment was determined. Cysteine 21 was predominantly labeled. Other labeled amino acids occurred at the NH2-terminal (Asn-2) and at position 26 (tryptophan). Due to the restricted mobility of the label in the lipid bilayer, these residues are suggested to be located in or close to the polar head of the lipid bilayer. These results will be compared with predictions for the arrangement of the polypeptide b derived from the hydrophobicity profile.     

Secondary structure composition of reconstituted subunit b of the Escherichia coli ATP synthase.

Subunit b of the Escherichia coli ATP synthase was isolated by preparative gel electrophoresis, acetone precipitated and after ion-pair extraction redissolved in a buffer either containing n-dodecyl-beta-D-maltoside or sodium cholate. The secondary structure of isolated subunit b was shown to be the same as within the FO complex, but was strongly dependent on the detergent used for replacement of the phospholipid environment. This was shown by an identical tryptic digestion pattern, which was strongly influenced by the detergent used for solubilization. An influence of the detergent n-dodecyl-beta-D-maltoside on the secondary structure of the hydrophilic part of subunit b was also shown for the soluble part of the polypeptide comprising residues Val25 to Leu156 (bsol) using CD spectroscopy. In order to determine the secondary structure of subunit b in its native conformation, isolated subunit b was reconstituted into E. coli lipid vesicles and analyzed with CD spectroscopy. The resulting spectrum revealed a secondary structure composition of 80% alpha helix together with 14% beta turn conformation. These results suggest that subunit b is not a rigid rod-like alpha helix simply linking F1 to FO, but rather provides an inherent flexibility for the storage of elastic energy within the second stalk generated by rotational movements within the F1FO complex.     

Fluidity of structure and swiveling of helices in the subunit c ring of Escherichia coli ATP synthase as revealed by cysteine-cysteine cross-linking

Subunit c in the membrane-traversing F(0) sector of Escherichia coli ATP synthase is known to fold with two transmembrane helices and form an oligomeric ring of 10 or more subunits in the membrane. Models for the E. coli ring structure have been proposed based upon NMR solution structures and intersubunit cross-linking of Cys residues in the membrane. The E. coli models differ from the recent x-ray diffraction structure of the isolated Ilyobacter tartaricus c-ring. Furthermore, key cross-linking results supporting the E. coli model prove to be incompatible with the I. tartaricus structure. To test the applicability of the I. tartaricus model to the E. coli c-ring, we compared the cross-linking of a pair of doubly Cys substituted c-subunits, each of which was compatible with one model but not the other. The key finding of this study is that both A21C/M65C and A21C/I66C doubly substituted c-subunits form high yield oligomeric structures, c(2), c(3)... c(10), via intersubunit disulfide bond formation. The results indicate that helical swiveling, with resultant interconversion of the two conformers predicted by the E. coli and I. tartaricus models, must be occurring over the time course of the cross-linking experiment. In the additional experiments reported here, we tried to ascertain the preferred conformation in the membrane to help define the most likely structural model. We conclude that both structures must be able to form in the membrane, but that the helical swiveling that promotes their interconversion may not be necessary during rotary function.     

Structural interactions between transmembrane helices 4 and 5 of subunit a and the subunit c ring of Escherichia coli ATP synthase

Subunit a plays a key role in promoting H+ transport and the coupled rotary motion of the subunit c ring in F1F0-ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F0 subunit c. H+ are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a. TMH4 of subunit a is thought to pack close to TMH2 of subunit c based upon disulfide cross-link formation between Cys substitutions in both TMHs. Here we substituted Cys into the fifth TMH of subunit a and the second TMH of subunit c and tested for cross-linking using bis-methanethiosulfonate (bis-MTS) reagents. A total of 62 Cys pairs were tested and 12 positive cross-links were identified with variable alkyl length linkers. Cross-linking was achieved near the middle of the bilayer for the Cys pairs a248C/c62C, a248C/ c63C, a248C/c65C, a251C/c57C, a251C/c59C, a251C/c62C, a252C/c62C, and a252C/c65C. Cross-linking was achieved near the cytoplasmic side of the bilayer for Cys pairs a262C/c53C, a262C/c54C, a262C/c55C, and a263C/c54C. We conclude that both aTMH4 and aTMH5 pack proximately to cTMH2 of the c-ring. In other experiments we demonstrate that aTMH4 and aTMH5 can be simultaneously cross-linked to different subunit c monomers in the c-ring. Five mutants showed pH-dependent cross-linking consistent with aTMH5 changing conformation at lower pH values to facilitate cross-linking. We suggest that the pH-dependent conformational change may be related to the proposed role of aTMH5 in gating H+ access from the periplasm to the cAsp-61 residue in cTMH2.     

The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism

Subunit a plays a key role in promoting H(+) transport and the coupled rotary motion of the subunit c ring in F(1)F(0)-ATP synthase. H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(0) subunit c. H(+) are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a based upon the chemical reactivity of Cys substituted into these helices. Here we substituted Cys into loops connecting TMHs 1 and 2 (loop 1-2) and TMHs 3 and 4 (loop 3-4). A large segment of loop 3-4 extending from loop residue 192 loop to residue 203 in TMH4 at the lipid bilayer surface proved to be very sensitive to inhibition by Ag(+). Cys-161 and -165 at the other end of the loop bordering TMH3 were also sensitive to inhibition by Ag(+). Further Cys substitutions in residues 86 and 93 in the middle of the 1-2 loop proved to be Ag(+)-sensitive. We next asked whether the regions of Ag(+)-sensitive residues clustered together near the surface of the membrane by combining Cys substitutions from two domains and testing for cross-linking. Cys-161 and -165 in loop 3-4 were found to cross-link with Cys-202, -203, or -205, which extend into TMH4 from the cytoplasm. Further Cys at residues 86 and 93 in loop 1-2 were found to cross-link with Cys-195 in loop 3-4. We conclude that the Ag(+)-sensitive regions of loops 1-2 and 3-4 may pack in a single domain that packs at the ends of TMHs 3 and 4. We suggest that the Ag(+)-sensitive domain may be involved in gating H(+) release at the cytoplasmic side of the aqueous access channel extending through F(0).     

Intrinsic uncoupling in the ATP synthase of Escherichia coli

The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 microM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126-11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi.     

F0 portion of Escherichia coli ATP synthase: orientation of subunit c in the membrane

Incubation of right-side-out oriented membrane vesicles of Escherichia coli with tetranitromethane resulted in the nitration of tyrosine residues (Tyr-10 and Tyr-73) of subunit c from the ATP synthase. Cleavage of the protein with cyanogen bromide and separation of the resulting fragments, especially of the tyrosine-containing peptides, clearly demonstrated that the distribution of the nitro groups is similar at any time and at any pH value chosen for the analysis. Furthermore, the percentage of 3-nitrotyrosine present in the two peptide fragments was in good agreement with that obtained for the intact polypeptide chain. While the modification of the tyrosine residues in subunit c with the lipophilic tetranitromethane is independent of the orientation of the membrane vesicles, the subsequent partial conversion of the 3-nitrotyrosine to the amino form only occurred when membrane vesicles with right-side-out orientation were treated with the ionic, water-soluble sodium dithionite, which at certain concentrations cannot penetrate biological membranes. Cleavage of subunit c isolated from nitrated and subsequently reduced membrane vesicles and separation of the resulting fragments by high-pressure liquid chromatography showed that the 3-nitrotyrosine in the Tyr-73-containing peptides has been completely reduced, while the nitro group in peptides containing Tyr-10 remained nearly unaffected.     

A model for the structure of subunit a of the Escherichia coli ATP synthase and its role in proton translocation

Most of what is known about the structure and function of subunit a, of the ATP synthase, has come from the construction and isolation of mutations, and their analysis in the context of the ATP synthase complex. Three classes of mutants will be considered in this review. (1) Cys substitutions have been used for structural analysis of subunit a, and its interactions with subunit c. (2) Functional residues have been identified by extensive mutagenesis. These studies have included the identification of second-site suppressors within subunit a. (3) Disruptive mutations include deletions at both termini, internal deletions, and single amino acid insertions. The results of these studies, in conjunction with information about subunits b and c, can be incorporated into a model for the mechanism of proton translocation in the Escherichia coli ATP synthase.     

Close proximity of a cytoplasmic loop of subunit a with c subunits of the ATP synthase from Escherichia coli

Interactions between subunit a and the c subunits of the Escherichia coli ATP synthase are thought to control proton translocation through the F(o) sector. In this study cysteine substitution mutagenesis was used to define the cytoplasmic ends of the first three transmembrane spans of subunit a, as judged by accessibility to 3-N-maleimidyl-propionyl biocytin. The cytoplasmic end of the fourth transmembrane span could not be defined in this way because of the limited extent of labeling of all residues between 186 and 206. In contrast, most of the preceding residues in that region, closer to transmembrane span 3, were labeled readily. The proximity of this region to other subunits in F(o) was tested by reacting mono-cysteine mutants with a photoactivated cross-linker. Residues 165, 169, 173, 174, 177, 178, and 182-184 could all be cross-linked to subunit c, but no sites were cross-linked to b subunits. Attempts using double mutants of subunit a to generate simultaneous cross-links to two different c subunits were unsuccessful. These results indicate that the cytoplasmic loop between transmembrane spans 3 and 4 of subunit a is in close proximity to at least one c subunit. It is likely that the more highly conserved, carboxyl-terminal region of this loop has limited surface accessibility due to protein-protein interactions. A model is presented for the interaction of subunit a with subunit c, and its implications for the mechanism of proton translocation are discussed.     

Proximity between periplasmic loops in the lactose permease of Escherichia coli as determined by site-directed spin labeling

Site-directed thiol cross-linking indicates that the first periplasmic loop (loop I/II) in the lactose permease of Escherichia coli is in close proximity to loops VII/VIII and XI/XII [Sun, J., and Kaback, H. R. (1997) Biochemistry 36, 11959-11965]. To determine whether thiol cross-linking reflects proximity as opposed to differences in the reactivity and/or dynamics of the Cys residues that undergo cross-linking, single-Cys mutants in loops I/II, VII/VIII, and XI/XII and double-Cys mutants in loop I/II and VII/VIII or XI/XII were purified and labeled with a sulfhydryl-specific nitroxide spin label. The labeled mutants were then analyzed by electron paramagnetic resonance (EPR) spectroscopy, and interspin distance was estimated from the extent of line shape broadening in the double-labeled proteins. Out of six paired double-Cys mutants that exhibit thiol cross-linking, five display significant spin-spin interaction. Furthermore, there is a qualitative correlation between distances estimated by site-directed cross-linking and EPR. Taken as a whole, the results are consistent with the conclusion that site-directed thiol cross-linking is primarily a reflection of proximity.     

The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli

The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches. First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function. This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis. Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122. Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected. Double mutants were constructed with a mutation that permits cross-linking to the b subunit. Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114. Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA. In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation.