The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship

The peripheral stalk of F1F0 ATP synthase is essential for the binding of F1 to FO and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b′ subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463–5471). The spatial arrangement of the chimeric b and b′ subunits, abbreviated Tb and Tb′, has been investigated by Cu2+-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F1F0 ATP synthase complexes. In both cases, disulfides were preferentially formed between TbA83C and Tb′A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist. The peripheral stalk of F1F0 ATP synthase is essential for the binding of F1 to FO and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b′ subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463–5471). The spatial arrangement of the chimeric b and b′ subunits, abbreviated Tb and Tb′, has been investigated by Cu2+-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F1F0 ATP synthase complexes. In both cases, disulfides were preferentially formed between TbA83C and Tb′A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist. F1F0 ATP synthases are found in the inner mitochondrial membrane, the thylakoid membrane of chloroplasts, and the cytoplasmic membrane of bacteria (1von Ballmoos C. Cook G.M. Dimroth P. Annu. Rev. Biophys. 2008; 37: 43-64Crossref PubMed Scopus (143) Google Scholar, 2Devenish R.J. Prescott M. Rodgers A.J. Int. Rev. Cell. Mol. Biol. 2008; 267: 1-58Crossref PubMed Scopus (79) Google Scholar, 3Nakamoto R.K. Scanlon J.A. Al-Shawi M.K. Arch. Biochem. Biophys. 2008; 476: 43-50Crossref PubMed Scopus (128) Google Scholar, 4Dimroth P. von Ballmoos C. Meier T. EMBO Rep. 2006; 7: 276-282Crossref PubMed Scopus (107) Google Scholar). These enzymes are responsible for harnessing an electrochemical gradient of protons across the membranes for the synthesis of ATP. In Escherichia coli F1F0 ATP synthase, the membrane-embedded F0 sector is composed of subunits ab2c10 and a soluble F1 portion composed of subunits α3β3γδϵ. The F0 sector houses a proton channel located principally in the a subunit, and the flow of protons through F0 generates torque used to rotate the c10 subunit ring relative to the ab2 subunits. The F1 γ and ϵ subunits are bound to the c10 ring and form the central or rotor stalk. Catalytic sites are located at the interfaces of each αβ pair in F1. The γ subunit extends into the center of the α3β3 hexamer, creating an asymmetry in the conformations of the αβ pairs (5Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2744) Google Scholar). It is the rotation of the γ subunit and the resulting sequential conformational changes in each αβ pair that provides the driving force for the synthesis of ATP at the catalytic sites. The α3β3 hexamer is held stationary relative to the rotary stalk by the peripheral stalk consisting of the b2δ subunits. The peripheral stalk is essential for binding F1 to F0 and for coupling proton translocation to catalytic activity (6Weber J. Biochim. Biophys. Acta. 2006; 1757: 1162-1170Crossref PubMed Scopus (52) Google Scholar, 7Weber J. Trends. Biochem. Sci. 2007; 32: 53-56Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 8Walker J.E. Dickson V.K. Biochim Biophys. Acta. 2006; 1757: 286-296Crossref PubMed Scopus (152) Google Scholar). In the E. coli enzyme, the peripheral stalk is a dimer of identical b subunits. The stalk has been conceptually divided into functional domains called the membrane domain (bM1-I33), the tether domain (bE34-A61), the dimerization domain (bT62-K122), and the F1-binding domain (bQ123-L156) (9Revington M. McLachlin D.T. Shaw G.S. Dunn S.D. J. Biol. Chem. 1999; 274: 31094-31101Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Although there is ample evidence of direct protein-protein interactions between b subunits within the membrane, dimerization, and F1-binding domains, there is remarkably little evidence of tight packing between the b subunits in the tether domain. In fact, electron spin resonance studies suggested that the tether domains of the two b subunits may be separated by more than 20 Å in the F1F0 complex (10Zaida T. Hornung T. Volkov O. Hoffman A. Pandey S. Wise J. Vogel P. J. Bioenerg. Biomembr. 2008; 40: 551-559Crossref PubMed Scopus (2) Google Scholar, 11Steigmiller S. Börsch M. Gräber P. Huber M. Biochim. Biophys. Acta. 2005; 1708: 143-153Crossref PubMed Scopus (44) Google Scholar). Much of what is known about the structure of the stalk has been inferred from analysis of the properties of polypeptides modeling segments of the b subunit. The structure of a peptide modeling the membrane domain, bM1-E34, has been determined by NMR (12Dmitriev O. Jones P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1999; 274: 15598-15604Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and a peptide based on the dimerization domain, bT62-K122, has been determined by x-ray diffraction (13Del Rizzo P.A. Bi Y. Dunn S.D. Shilton B.H. Biochemistry. 2002; 41: 6875-6884Crossref PubMed Scopus (87) Google Scholar). Both polypeptides assumed α-helical conformations, but neither structure directly revealed b subunit dimerization interactions. Recently, Priya et al. (14Priya R. Tadwal V.S. Roessle M.W. Gayen S. Hunke C. Peng W.C. Torres J. Grüber G. J. Bioenerg. Biomembr. 2008; 40: 245-255Crossref PubMed Scopus (7) Google Scholar) reported a low resolution structure of a bM22-L156 dimer, but the extended conformation appears to be slightly too long to accurately reflect the dimensions of the peripheral stalk within the F1F0 complex. Molecular modeling efforts supported by a variety of biochemical and biophysical experiments have yielded competing right-handed coiled coil (15Wood K.S. Dunn S.D. J. Biol. Chem. 2007; 282: 31920-31927Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 16Del Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar) and coiled coil T. Volkov S. Wise Vogel Biophys. J. 2008; Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Vogel Biophys. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) for the peripheral stalk. The parallel coiled coli is a known structure by packing of the of the two helices that are conformation that on the b a to in the b subunit. In contrast, Rizzo et al. Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar) a parallel right-handed coiled coil with the helices of the two b subunits by and a of an staggered model positions the two identical by each of the b subunits in a into and at from have been in of both Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar, Vogel Biophys. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). In of studies of polypeptides have evidence that dimer packing be at sites from and to the in model and in (9Revington M. McLachlin D.T. Shaw G.S. Dunn S.D. J. Biol. Chem. 1999; 274: 31094-31101Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 16Del Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar). at a of the positions has been within intact F1F0 ATP synthase A.J. S. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). electron spin resonance on have also been for the arrangement T. Volkov S. Wise Vogel Biophys. J. 2008; Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Vogel Biophys. J. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). with polypeptides modeling the b subunit has generated evidence a staggered conformation in this of the dimer (15Wood K.S. Dunn S.D. J. Biol. Chem. 2007; 282: 31920-31927Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 16Del Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar, D.T. Dunn S.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In of dimerization domain polypeptides with at disulfides preferentially formed between positions that were were into the conformation by the formation of disulfide between at positions and and (15Wood K.S. Dunn S.D. J. Biol. Chem. 2007; 282: 31920-31927Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). These staggered were more with and bound soluble F1 with than in the and F1F0 were with heterodimeric peripheral stalks b subunits with tether domains in by J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These F1F0 had peripheral stalks that were by of at within the tether domain. In to the of identical b subunits in the peripheral stalk of E. photosynthetic organisms express two b-like subunits, b and that are to form heterodimeric peripheral stalks in F1F0 ATP we generated heterodimeric peripheral stalks within the E. coli F1F0 by chimeric b subunits Dunn S.D. J. Bacteriol. 2007; PubMed Scopus Google Scholar). of the tether and dimerization domains of the E. coli b subunit were replaced with the homologous of the Thermosynechococcus elongatus b and b′ subunits. The chimeric subunits formed heterodimeric peripheral stalks that were into functional F1F0 ATP synthase complexes. The chimeric enzymes had T. elongatus sequences of the E. coli b subunit. these chimeric subunits be to Tb and The to F1F0 ATP synthases with heterodimeric peripheral stalks a to the positions of the two subunits in the peripheral stalk. In the we that the Tb and subunits assumed positions relative to within the F1F0 complex. The staggered conformation appears to be a and functional conformation for the peripheral stalk. within a of F1F0 with peripheral stalks in the conformation are to exist. The for membrane was an E. coli that a of the J. Bacteriol. PubMed Google Scholar). was used for the of modeling the b subunit M. R. Nature. PubMed Scopus Google Scholar). F1F0 ATP synthase was in by on with The for membrane were in with and and were of the were at b subunit was by that a and a on the The is in and subunit but be in the subunit the for was generated by the subunit from into the sites of were into b subunits by of the in The were for the of chimeric Tb or subunits with a for or Tb subunit were in that has T. elongatus b subunit for and a at the were based on that has the T. elongatus b′ subunit for the and a on the of the were by direct of E. coli b subunit with from the b subunit of T. elongatus for a and a at the E. coli b subunit with from the b′ subunit of T. elongatus for a and a at the at been to in and genes modeling the dimerization domains of the Tb and subunits. These have the at the the and with the at the and genes modeling the of the Tb and subunits. These also with the and have at or on than no or J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Dunn S.D. J. Bacteriol. 2007; PubMed Scopus Google Dunn S.D. J. Bacteriol. 2007; PubMed Scopus Google Dunn S.D. J. Bacteriol. 2007; PubMed Scopus Google E. coli b subunit with from the b subunit of T. elongatus for a and a at the Tb′, E. coli b subunit with from the b′ subunit of T. elongatus for a and a at the The at been to in The and genes modeling the dimerization domains of the Tb and subunits. These have the at the the and with the at the The and genes modeling the of the Tb and subunits. These also with the and have at or than no in a were also to of soluble polypeptides on the Tb and subunits of from the were by the in and into the S.D. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the and sites. The for of the Tb and the an and a with by Tb or at were to express modeling the Tb or subunit by of and were used the and the to and abbreviated and modeling the Tb and dimerization domain and a on the were the and or the The resulting and are abbreviated and was into the R. T. PubMed Scopus Google Scholar) the and sites to and with were R.K. PubMed Scopus Google Scholar). were determined the Biochem. PubMed Scopus Google Scholar). ATP-driven proton were by of membrane to of reaction used and the of Biochemistry. 37: PubMed Scopus Google Scholar). was a The ATP in membrane were determined the Sci. S. A. PubMed Scopus Google Scholar). the of ATP by the to the reaction of with to form The of was an at a was by the in to a of and at was in to a of and in at The was to and both and were on reaction of of membrane of of of and to a of The were in a to and into a to reaction was to for to the by the of of ATP in were for The of the portion of the reaction was determined in of the with the of ATP by was determined by to each reaction to a of Arch. Biochem. Biophys. 1999; PubMed Scopus Google Scholar). peripheral stalks were a J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were and a to F1F0 ATP synthase at and were separated on a for analysis J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). used were an the b subunit from G. and an the used were and The of bound was by with the The chimeric dimerization domains and were by in both and The Tb and were separated by in The were by to that have been Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar) and by that with at between and of were on at separated the domains into two of the by on at yielded Tb dimerization domain. of the to the in b′ on a of yielded the polypeptides consisting of and at or were by of or and of the by the were to with The were by and and These were used for disulfide formation studies The and of each was by the of the Tb and subunits by of the to a of to the in a were determined by the Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar). were on a (9Revington M. McLachlin D.T. Shaw G.S. Dunn S.D. J. Biol. Chem. 1999; 274: 31094-31101Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Disulfide between b subunits in the peripheral stalk were formed by membrane with for for each The were in with at in an to reaction was by to a of through and on were for each a and a or The was by the membranes to in and to with and The reaction was and to at for and on for or was by the membrane to in and to in the was and the were to with for or was by the of of of each cross-linked was by an the in the was the The of the soluble Tb and dimerization domains with and to form and was determined by of or an of the Tb and at and at of or The was in each The were with to cross-link formation. The of chimeric polypeptides consisting of and at or to form disulfide was by of or into a by the of and to at for the were with to cross-link formation. a b subunit with a was generated to and of subunits. for evidence of an conformation in the dimerization domain of the peripheral stalk of the E. coli F1F0 ATP synthase, were within the for of subunits and These positions were based on with model polypeptides Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar). of the was into E. coli of the was by on with The experiments that b subunits were into F1F0 ATP synthase it be that the of the were than a with the to of the had no effect on The membranes from each were and studied for ATP activity in both the of of a of the relative of intact F1F0 from had of the activity in the but in the membrane were from for The of coupling between ATP at F1 and proton at F0 was a ATP-driven proton Although of activity were in of the the b subunit membranes had proton activity. be membranes the with of activity found in b subunits supported complex and detectable of F1F0 ATP synthase activity of for in of of membrane to the effect of for in of of membrane to the effect of in a The to for an conformation for b subunits within the peripheral stalk was In these E. coli b subunits with the were a peripheral stalk. Disulfide cross-link formation was by membranes from subunits or for with of the of and cross-linked subunits were determined by analysis an the of experiments revealed that of the subunits be of the and subunits were and little cross-link formation was detectable in the The evidence of the existence of an conformation of the peripheral stalk in intact F1F0 ATP the disulfide were of under more conditions, the was to and the reaction was to dimer formation was with at or the cross-link in the peripheral stalk was detectable under more stringent reaction in the E. coli b subunits evidence an model for the peripheral but the staggered model be in a stalk the two b subunits are from each we the heterodimeric chimeric subunit A.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to a of the staggered conformation the properties of Tb and with the on polypeptides modeling the E. coli b subunit, the dimerization domains of the Tb and subunits were and to of both and of at and that the chimeric form both and formation of was the dimerization domains were with Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar) the E. coli dimerization domain that disulfide formation between the the and of the Disulfide formation were on the polypeptides and Cu2+-mediated in the of of to In the of both domains formed disulfides The of the domains but the dimer was from the dimer by a in The with an of the two domains an and was the The of dimer formation in this be by the of to the that are located to a are more to a disulfide with the The of the of formation in the These that each of the chimeric was of under the conditions of the but that the formation of was in of the T. elongatus portion of the The of disulfide formation between for and was soluble of the chimeric subunits that of were or for in a and by Both the and polypeptides and the of both to form no dimer formation was for of the with at the possible dimer formation was found to be the in the a for of and of formation for the peptide was Importantly, the T. elongatus sequences to be of the chimeric relative to were to express the chimeric and subunits. The chimeric subunits were in and in and for in F1F0 activity by on the of the subunits the and supported formation with the chimeric subunits The membranes were from the chimeric subunit and for ATP activity in both the and the of of and chimeric subunits yielded membrane with found in of coupling the proton that the subunits no effect on coupling The of disulfide formation in F1F0 ATP synthase peripheral stalks were in membranes from the both chimeric Tb and subunits. The experiments were by under conditions and was in membranes chimeric subunit and of the was each a of F1F0 with heterodimeric stalks and and peripheral from a pair of chimeric subunits of subunit a a of cross-link formation indicating that peripheral stalks to the cross-linked in the membrane both subunits to detect cross-link formation to heterodimeric peripheral each was a and by an the dimer formation was for both the and peripheral The be with to an conformation or an staggered conformation but for both competing It be that the cross-linked peripheral stalk was detectable in with the The was in with the of the in the the evidence that the chimeric subunits and positions within the heterodimeric The evidence a staggered conformation within the dimerization domain that has the Tb subunits in the with to the subunit. The was to to more form that with the of F1F0 but for direct of enzymatic activity. Under these conditions the that was cross-linked peripheral The dimer was the cross-linked of F1F0 complex and in a a that be from the of dimer formation in and subunits or and subunits. reaction that the within analysis of the that of the subunit was with a TbA83C subunit and cross-linked with the a of cross-link formation in the of the the was on an structure in the peripheral and less than of the peripheral stalks were in a staggered than it were to to an conformation for of the in the by disulfide formation be to have a detectable on activity. The effect of cross-link formation on activity was the proton Importantly, the formation of this cross-link had no effect on that a peripheral stalk in the arrangement a functional form of the enzyme. The of this was to direct of two competing of the peripheral stalk of F1F0 ATP model that the b subunits are in a staggered parallel conformation (15Wood K.S. Dunn S.D. J. Biol. Chem. 2007; 282: 31920-31927Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 16Del Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar, D.T. Dunn S.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and the suggested an parallel arrangement (9Revington M. McLachlin D.T. Shaw G.S. Dunn S.D. J. Biol. Chem. 1999; 274: 31094-31101Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, A.J. S. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have evidence compatible with both conformations, but the staggered positions to be The model was supported by the of disulfide formation in E. coli peripheral stalks both b subunits had identical Disulfide cross-link formation was at sites within the dimerization domain and it was possible to a to the model the E. coli peripheral stalk both b subunits are identical and within the this we Tb and chimeric subunits by of the E. coli b subunit with a homologous from the T. elongatus Dunn S.D. J. Bacteriol. 2007; PubMed Scopus Google Scholar). The the arrangement were in F1F0 and chimeric subunits. studies both polypeptides modeling the chimeric subunits and the chimeric subunits into intact functional F1F0 ATP synthases that the chimeric subunits positions relative to by the T. elongatus was in the heterodimeric peripheral a staggered conformation with the Tb subunit in the relative to the Rizzo P.A. Bi Y. Dunn S.D. J. Mol. Biol. 2006; 364: 735-746Crossref PubMed Scopus Google Scholar), these that in the heterodimeric stalk of the b′ subunit preferentially the and the b subunit the The dimer formation within that the arrangement of the heterodimeric peripheral stalks is the staggered the in have on the activity of F1F0 the staggered parallel conformation is a functional structure for the peripheral stalk. The evidence from is to be in of a or right-handed coiled coil in the peripheral stalk. Disulfide cross-link formation by of evidence of the existence of an conformation in both chimeric and peripheral The cross-link was formed between at in both cases, and of the was cross-linked to in peripheral stalks in the staggered conformation but a of of the and peripheral stalks were also cross-linked in the of a or right-handed coiled and be located less than Å in the that the disulfide is be within of both TbA83C and model the peripheral stalk in between the staggered and conformations the that both be the that the staggered arrangement be with less that this more cross-link the is the conformations the chimeric subunit enzymes are of conformations in F1F0 ATP are of evidence that this the Tb and subunits were into the E. coli F1F0 ATP synthase in Dunn S.D. J. Bacteriol. 2007; PubMed Scopus Google Scholar). the chimeric enzymes ATP-driven proton which that the chimeric subunits be into F1F0 but also be that in of the protein-protein interactions within both F1 and F0 for a functional complex. the for the E. coli and peripheral stalks are identical to of the chimeric and peripheral suggesting that the conformation of the chimeric peripheral stalk may be the the peripheral stalk. it that the is that the properties of the chimeric Tb and subunits accurately reflect the normal of a b subunit. for the staggered conformation experiments in a photosynthetic with b and b′ subunits. are to the of with

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