The b and δ Subunits of the Escherichia coli ATP Synthase Interact via Residues in their C-terminal Regions

Abstract

An affinity resin for the F1sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader.b 24–156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms ofb 24–156, in which one or four residues from the C terminus were removed, competed poorly for F1binding, suggesting that these residues play an important role inb -F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24–156 resulted in a disruption of its association with the purified δ subunit of the enzyme. To determine whether these residues interact directly with δ, cysteine residues were introduced at various C-terminal positions ofb and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and δ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24–156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue δ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that theb and δ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0. An affinity resin for the F1sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader.b 24–156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms ofb 24–156, in which one or four residues from the C terminus were removed, competed poorly for F1binding, suggesting that these residues play an important role inb -F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24–156 resulted in a disruption of its association with the purified δ subunit of the enzyme. To determine whether these residues interact directly with δ, cysteine residues were introduced at various C-terminal positions ofb and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and δ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24–156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue δ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that theb and δ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0. In the process of oxidative phosphorylation or photophosphorylation, the electron transport chain generates a transmembrane proton gradient. The ATP synthase, or F1F0-ATPase, allows protons to flow down this electrochemical gradient and uses the energy obtained to synthesize ATP (for reviews, see Refs. 1Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar, 2Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 3Deckers-Hebestreit G. Altendorf K. Annu. Rev. Microbiol. 1996; 50: 791-824Crossref PubMed Scopus (170) Google Scholar, 4Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (395) Google Scholar). Under appropriate conditions ATP synthase can hydrolyze ATP to pump protons. The enzyme is composed of two sectors; the F0 sector is membrane-integral and is responsible for proton translocation, and the F1 sector is attached to the membrane via F0 and houses the catalytic sites for ATP synthesis. F1 is easily detached from the membrane and can be purified as a soluble protein with ATPase activity. ATP synthases contain at least eight types of subunits. In the relatively simple enzyme from Escherichia coli , the F1 sector has the stoichiometry α3β3γ1δ1ε1, whereas F0 is composed of three subunits of stoichiometrya 1 b 2 c 9–12. The a and c subunits, but not b , contain residues essential for the translocation of protons across the membrane (3Deckers-Hebestreit G. Altendorf K. Annu. Rev. Microbiol. 1996; 50: 791-824Crossref PubMed Scopus (170) Google Scholar). The 156-residue b subunit is believed to span the membrane once at its hydrophobic N terminus, whereas the remainder of the protein is very hydrophilic. b is thought to exist as a dimer in the complex (5Schairer H.U. Hoppe J. Sebald W. Friedl P. Biosci. Rep. 1982; 2: 631-639Crossref PubMed Scopus (7) Google Scholar, 6Aris J.P. Simoni R.D. J. Biol. Chem. 1983; 258: 14599-14609Abstract Full Text PDF PubMed Google Scholar, 7Hermolin J. Gallant J. Fillingame R.H. J. Biol. Chem. 1983; 258: 14550-14555Abstract Full Text PDF PubMed Google Scholar), and proteolysis studies have shown that the hydrophilic region of b is required for the association of F1 with the membrane (7Hermolin J. Gallant J. Fillingame R.H. J. Biol. Chem. 1983; 258: 14550-14555Abstract Full Text PDF PubMed Google Scholar, 8Hoppe J. Montecucco C. Friedl P. J. Biol. Chem. 1983; 258: 2882-2885Abstract Full Text PDF PubMed Google Scholar, 9Perlin D.S. Cox D.N. Senior A.E. J. Biol. Chem. 1983; 258: 9793-9800Abstract Full Text PDF PubMed Google Scholar). Removal of two residues from the C terminus of b disrupts normal assembly of the complex (10Takeyama M. Noumi T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 16106-16112Abstract Full Text PDF PubMed Google Scholar), as does mutation of Gly-131 to aspartate (11Jans D.A. Hatch L. Fimmel A.L. Gibson F. Cox G.B. J. Bacteriol. 1985; 162: 420-426Crossref PubMed Google Scholar). Thus theb subunit is essential for linking the F1 and F0 sectors and likely plays a key role in the coupling of energy from proton translocation to ATP synthesis. The crystal structure of the mitochondrial F1 has shown that the α and β subunits alternate in a hexagonal ring structure, with two long α-helices from γ extending into a hole in the center of the ring (12Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2754) Google Scholar). Recent evidence strongly suggests that γ and probably ε rotate relative to α and β during catalysis (13Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (459) Google Scholar, 14Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar, 15Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar, 16Aggeler R. Ogilvie I. Capaldi R.A. J. Biol. Chem. 1997; 272: 19621-19624Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Several studies have implied that the δ subunit is located near the top of the αβ cluster (17Dunn S.D. Heppel L.A. Fullmer C.S. J. Biol. Chem. 1980; 255: 6891-6896Abstract Full Text PDF PubMed Google Scholar, 18Maggio M.B. Parsonage D. Senior A.E. J. Biol. Chem. 1988; 263: 4619-4623Abstract Full Text PDF PubMed Google Scholar, 19Lill H. Hensel F. Junge W. Engelbrecht S. J. Biol. Chem. 1996; 271: 32737-32742Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 20Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Like b , δ is required for the binding of F1 to F0. Membranes of mutant E. coli strains expressing truncated forms of δ showed little ATPase activity (21Jounouchi M. Takeyama M. Chaiprasert P. Noumi T. Moriyama Y. Maeda M. Futai M. Arch. Biochem. Biophys. 1992; 292: 376-381Crossref PubMed Scopus Google Scholar), suggesting that to the membrane in the of In and studies the mitochondrial S. C. J. 1996; PubMed Scopus Google Scholar, S. C. J. 1997; PubMed Scopus Google and Y. Arch. Biochem. Biophys. 1997; PubMed Scopus Google of δ, b form of the b subunit residues to with the N-terminal b and b forms of the b subunit the residues with the N-terminal or form of the b subunit to b 24–156 but with and to the C chain the C-terminal region of was in F0 The subunit of F0 able to span the from the membrane to the top of F1 is b The hydrophilic of b is has an and to F1 S.D. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). cross-linking of E. coli ATP synthase has to have obtained with the G. H. Biochim. Biophys. Acta. 1992; PubMed Scopus Google and mitochondrial S. R. J. Biol. Chem. Full Text PDF PubMed Google In the the by was to the C-terminal of b The site in δ was to be within the residues to of the Recent studies Walker J.E. J. Biol. 1994; Google Scholar, K. H. C. H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Aggeler R. M.B. Capaldi R.A. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have the interaction of E. coli b and δ in the of subunits. In the the interaction between E. coli b and F1. An F1 affinity resin has by linking the hydrophilic of b to a solid binding this analytical and have evidence to an interaction between residues at the C terminus of b and the C-terminal region of were as by J. T. Scholar). which was as an during the of was from S.D. 1996; PubMed Scopus Google by of site of the followed by of the from S.D. J. Biol. Chem. 1992; Full Text PDF PubMed Google into the and The was that the is of the b was as S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google was as the for the the an site and the is to the of the b The is The for was the sequencing The was into the and sites of to a protein with the followed by residues through of the b The was to that mutation during the expressing of b b and the and were S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In were to the mutation and were into unique of the into of the was by To into the b appropriate were to the from the and to S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The of b S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). b 24–156 were and purified as S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). and truncated forms ofb 24–156 as as b and purified in the 24–156, that cysteine residues were purified in the of 1 or b were into the E. coli J. Bacteriol. PubMed Google Scholar). of the and of were as S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The δ subunit was and purified as S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). F1 was purified by S.D. Heppel L.A. J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar). was from b by through a with 1 obtained from was with of of of b of The was at with for 1 and was and for the of protein in the the ofb to the resin was to be of The resin was at in 1 cysteine to 1 was the resin was with and was to its in at The resin was at resin was prepared by the with 1 cysteine of the b for F1 binding were In the ATPase of F1 with of the δ subunit was with of the affinity resin in a of of 2 and The δ subunit was to the to for of that subunit in the a of δ is easily during of the complex M. Heppel L.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). for 1 at the was to the The ATPase activity of the was as M. Heppel L.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the δ, and the affinity resin in the were in of the binding the and the was and the was in of at for the was to the and of the were by ultracentrifugation was a at and 1 was In velocity the was and were at of δ, 1 was to the the during in which δ and forms of b were The were with the the of Biochem. 1992; Scholar). 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The and were of Altendorf and of was purified δ subunit PubMed Scopus Google Scholar), and the were affinity purified a δ S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were by the Biochem. Biophys. PubMed Scopus Google Scholar). were by the of Biochem. PubMed Scopus Google or A.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). sequencing was at the for at an To the binding between the F1 sector and the hydrophilic of b , this region of b to a solid to form an affinity resin for F1. The coupling from to which can be via was as a the hydrophilic region of b has cysteine the site of coupling to the resin can be by of b the C-terminal region was thought likely to be inb -F1 introduced a cysteine residue near the N terminus of the hydrophilic the was as The four of this were from the E. coli enzyme of the of the and the of this enzyme in a K. Y. Y. S. S. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The purified b was to the resin as at a of of of the modified resin with the F1 complex followed by a of F1 with the as by a of F1 with the resin that modified with cysteine 1 The F1 in this was probably not to the resin but was between and within the resin a for the of was in of were to with both conditions Thus the b resin is able to F1 The at the of b 24–156 in the a of b from the resin during the in likely this from in which one subunit of the dimer to the To the of the hydrophilic ofb to compete with the resin for binding to the resin was with F1 in the of of b 24–156 as b S.D. 1996; PubMed Scopus Google Scholar). and of the by the of F1 to the resin was to as the ofb 24–156 F1 was by the resin at ofb 24–156 dimer 1 that the of soluble b 24–156 within the resin an of the of These 24–156 is able to compete with theb resin for binding to a simple for the relative affinity of mutant form of b for F1. were b b which one and four residues from the C terminus, was that these forms ofb competed very relative 24–156, with theb resin 1 a dimer of showed a of whereas at the b by the is that very is to in this as a in To determine and in a the was modified that soluble ATPase ATPase was Under the conditions in these of the enzyme was by the for F1 binding 24–156, b was by the in ATPase activity in the when these forms of b were to the the of F1 in the with of b 24–156, whereas the of b to compete with theb resin was These results that the C-terminal residues of b essential for its interaction with The of soluble b were directly for ATP activity to that with an enzyme as not for the in soluble ATPase activity. In a for of the soluble activity. Recent ultracentrifugation results from this have evidence for the of an complex by two of the hydrophilic region ofb and one of the δ subunit S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to determine whether the binding of the C-terminal of b to be to of with The 24–156 and δ subunits showed which were to for b S.D. J. Biol. Chem. 1992; Full Text PDF PubMed Google and δ S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in the of of b δ is that these form a complex in of δ with b or resulted in the of the two that binding of b to δ is by removal of one or four residues from the C Thus the essential of these residues for binding F1 from their role in binding The ofb was by as to be that the ofb was by the C-terminal 24–156 S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), ofb was at velocity of δ with soluble b of three as were by ultracentrifugation at as was at a dimer of and δ was at of three S.D. in a as were by ultracentrifugation at as was at a dimer of and δ was at The protein b which residues of the b relative to b 24–156, was to have a 24–156 the complex when b was with δ a that ofb 24–156 and was in with the of for the b complex with theb that residues of b S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). that removal of residues near the N terminus of b have a its binding to δ, and the ofb 24–156 and b by that the of residues which in the the of theb complex to its is of that the ofb and b that ofb 24–156, which is a residues in that the truncated forms have b 24–156, probably to a in the C-terminal region of the C-terminal a be responsible for the binding of these forms of b to To between and of the C terminus of b in binding δ, to to δ from sites within this cysteine residues were introduced into b at positions and was that a to two and attached to the C terminus of b was that the to the C-terminal the of a these forms of b were able to for with as the that theb -F1 interaction was not in the F1F0 the b subunits were with the cross-linker and to The were by b as cross-linking was with b or b not of the were with both b and b The were the for a b and showed with that between b and Membranes F1F0 showed of activity with with or not To the the and were incorporated into b of of these with with and to of an appropriate were The were by b and δ and the of of the and to in the of F1 an in the of protein the and of revealed a of and which were probably not the of their ofb via 158 to purified F1. An to that in the to was that the mutation was of the of the soluble forms of b to a in that a b 24–156 and These were by and suggesting that in an inb was not to the in the of F1. of were with both the and the and In one of these was by the whereas the was not and In the of have one of these as a The was by an not and to To the region of δ in the 24–156, a the from cysteine mutation was from an and with as CNBr of b 24–156 to of and with the C-terminal to the site of the The δ subunit to between and residues in The 24–156 and its CNBr is CNBr of to a that is the from b 24–156 by in a the to a the CNBr from was from the membrane and by In both to two to and δ one of was the of b The the of δ the δ in both the and the residue that δ not at this by the was not in the appropriate of the of of the sequencing of cross-linking from The of the residue of the is in the sequencing a residue that not be b δ the of the of residues and of δ was to the of and the of from b δ were with and by as and the from J.E. M. Biochem. J. PubMed Scopus Google The of the residue of the is in the sequencing a residue that not be the of the of residues and of δ was to the of and the of from in a were with and by as and the The results that the residues at the C terminus of b essential for interaction with that the interaction of b with δ is when these residues and that the C-terminal region of b is to that of these subunits believed to form a from the membrane to near the top of the F1 which to the subunits during of γ and ε by proton flow through F0 W. D. Engelbrecht S. Chem. 1996; Google Scholar, S. S.D. J. Capaldi R.A. Biol. 1997; PubMed Scopus Google Scholar). and studies showed that the C terminus of b was essential for assembly of ATP synthase (10Takeyama M. Noumi T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 16106-16112Abstract Full Text PDF PubMed Google and implied that the C terminus of δ a role in interaction of F1 with the (21Jounouchi M. Takeyama M. Chaiprasert P. Noumi T. Moriyama Y. Maeda M. Futai M. Arch. Biochem. Biophys. 1992; 292: 376-381Crossref PubMed Scopus Google Scholar, S. C. J. 1996; PubMed Scopus Google Scholar, S. C. J. 1997; PubMed Scopus Google Scholar, Y. Arch. Biochem. Biophys. 1997; PubMed Scopus Google Scholar). studies and this by the b δ The site of cross-linking of the and to δ was shown to be C-terminal to is that the δ subunit was not at by and that to this residue was during the appropriate of peptide The of these results is that the site of was through the chain of Met-158. The have a at of this to a center that be relatively to by is that to or to G. 1994; PubMed Scopus Google Scholar). of the C-terminal region of b as an an structure The cross-linking results that the hydrophobic of this one of the b subunits with that the hydrophobic of the b subunit is in with region of b Removal of one or hydrophobic residues this a in the soluble b a to a the of b were that for b 24–156 In the of δ, the hydrophobic of the b subunit not have its normal and interact with the hydrophobic of 24–156 the of b 24–156 during at S.D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google is not with b residues at positions coli residues and in the b subunit of A. L. J. 1996; PubMed Scopus Google Scholar). is to that the of these hydrophobic residues in the b interaction the for the that disrupts the binding of F1 to F0. regions of b be inb -F1 was able to compete to a with the affinity resin for binding to F1 In this is that have b subunits that the hydrophobic residues at the C the b subunit of the at the residue to in E. coli b S. M. M. H. T. Y. K. Y. Biochim. Biophys. Acta. 1988; PubMed Scopus Google Scholar). The in between the and the was is that in the N-terminal region of the soluble b allows the hydrophobic residues inb 24–156 but inb to and interact with an be and the complex the N-terminal were in a the N of the b subunits in the membrane in ATP synthase, that residues of b interact with δ in the complex. Takeyama and (10Takeyama M. Noumi T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 16106-16112Abstract Full Text PDF PubMed Google showed that one of the ATP synthase assembly by of residues from the C terminus of b was the of F0 to form a proton in In and E. J. Bacteriol. PubMed Google Scholar, R.A. J. Bacteriol. 1994; PubMed Google that the δ subunit was required for the of the proton from the have shown that the region of b in the F0 assembly process is essential for binding to δ, the that the b interaction is for the assembly of F0. have evidence of a from the interaction be from the C-terminal ofb to the interaction with the F0 subunits The structure of residues of a of residues of δ, has by but the structure of the 177-residue subunit not be S. S.D. J. Capaldi R.A. Biol. 1997; PubMed Scopus Google Scholar). the C-terminal regions of both to be in the of from region is to between residues inb and The site of a C-terminal residues S. J. F. Junge W. PubMed Scopus Google Scholar). results that b and δ interact via their C-terminal and a from has shown that the b complex is to span the from the membrane to the N-terminal of α S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus is with the that theb and δ subunits form a to from relative to the an H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar), and of the b interaction is relatively S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), is likely that the interaction of these subunits is by the of the of the ATP synthase complex. is with subunits, as α or to the b -F1 The for b a and of in b -F1 affinity with this to residues of b that -F1 interactions. and Altendorf for the and and for coli for and of for and for