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• W2010071674 abstract "The structure of the N-terminal transmembrane domain (residues 1–34) of subunit b of theEscherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4–22 form an α-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23–26 with α-helical structure resuming at Pro-27 at an angle offset by 20° from the transmembrane helix. In native subunitb, the hinge region and C-terminal α-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2–21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an α-helix, over the span of residues 2–18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane α-helices are positioned at a 23° angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains. The structure of the N-terminal transmembrane domain (residues 1–34) of subunit b of theEscherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4–22 form an α-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23–26 with α-helical structure resuming at Pro-27 at an angle offset by 20° from the transmembrane helix. In native subunitb, the hinge region and C-terminal α-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2–21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an α-helix, over the span of residues 2–18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane α-helices are positioned at a 23° angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains. During oxidative and photo phosphorylation ATP is synthesized by a H+-transporting F0F1-ATP synthase. In mitochondria, chloroplasts, and eubacteria, the enzyme consists of an H+-transporting transmembrane domain, termed F0, and a catalytic domain bound at the membrane surface, termed F1. Each sector is composed of multiple subunits that vary somewhat between species (1Senior A.E. Physiol. Rev. 1988; 68: 177-231Crossref PubMed Scopus (461) Google Scholar). The simplest enzyme is found inE. coli where the composition is α3β3γ1δ1ε1for F1 anda 1 b 2 c 12for F0 (2Fillingame R.H. J. Exp. Biol. 1997; 200: 217-224Crossref PubMed Google Scholar, 3Jones P.C. Fillingame R.H. J. Biol. Chem. 1998; 273: 29701-29705Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The structure of much of the α3β3γ segment of F1 from bovine mitochondria has been solved by x-ray crystallography (4Abrahams J.P. Leslie A.G.W. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2754) Google Scholar) and dramatic progress made in understanding the mechanism of ATP synthesis by a binding change mechanism involving rotary catalysis (5Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1595) Google Scholar, 6Boyer P.D. Angew. Chem. Int. Ed. Engl. 1998; 37: 2296-2307Crossref PubMed Scopus (147) Google Scholar, 7Walker J.E. Angew. Chem. Int. Ed. Engl. 1998; 37: 2308-2319Crossref PubMed Scopus (248) Google Scholar). The γ subunit has been shown to rotate within a hexameric ring of α3β3 subunits to promote changes in substrate and product binding affinities at alternating catalytic sites within the β subunits (8Noji H. Yasuda R. Yoshida M. Kiniosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1966) Google Scholar, 9Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (459) Google Scholar, 10Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (464) Google Scholar). The mechanism by which proton translocation through F0 is coupled to rotary catalysis in F1 remains to be elucidated. Subunit c is believed to play the central role in proton transport via protonation-deprotonation of an essential Asp-61 carboxylate from alternate access channels on either side of the membrane (2Fillingame R.H. J. Exp. Biol. 1997; 200: 217-224Crossref PubMed Google Scholar). The structure of subunit c, the smallest subunit in F0, has been solved by heteronuclear NMR (11Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 37: 8817-8824Crossref PubMed Scopus (272) Google Scholar), and a ring-like organization of the c oligomer in F0 was recently elucidated by cross-linking approaches (12Jones P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1998; 273: 17178-17185Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar,13Fillingame R.H. Jones P.C. Jiang W. Valiyaveetil F.I. Dmitriev O.Y. Biochim. Biophys. Acta. 1998; 1365: 135-142Crossref PubMed Scopus (59) Google Scholar). Low resolution electron microscopic and atomic force microscopic images also suggest a ring-like arrangement of the coligomer with subunits a and b lying at the periphery of the ring (14Birkenhäger R. Hoppert M. Deckers-Hebestreit G. Mayer F. Altendorf K. Eur. J. Biochem. 1995; 230: 58-67Crossref PubMed Scopus (129) Google Scholar, 15Takeyasu K. Omote H. Nettikadan S. Tokumasu F. Iwamoto-Kihara A. Futai M. FEBS Lett. 1996; 392: 110-113Crossref PubMed Scopus (117) Google Scholar, 16Singh S. Turina P. Bustamante C.J. Keller D.J. Capaldi R.A. FEBS Lett. 1996; 397: 30-34Crossref PubMed Scopus (105) Google Scholar). The placement of subunits aand b at the outside of the ring is supported by cross-linking studies (13Fillingame R.H. Jones P.C. Jiang W. Valiyaveetil F.I. Dmitriev O.Y. Biochim. Biophys. Acta. 1998; 1365: 135-142Crossref PubMed Scopus (59) Google Scholar, 17Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar). To couple H+ transport to rotary catalysis in F1, H+-flux through F0 is proposed to drive rotation of the coligomeric ring relative to the stationary subunits a andb at the periphery of the complex (9Duncan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (459) Google Scholar, 18Vik S.B. Antonio B.J. J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar, 19Engelbrecht S. Junge W. FEBS Lett. 1997; 414: 485-491Crossref PubMed Scopus (118) Google Scholar, 20Elston T. Wang H. Oster G. Nature. 1998; 391: 510-513Crossref PubMed Scopus (446) Google Scholar). In such a model, subunit b is proposed to play the role of a stator, holding the α3β3 subunits of F1fixed to the stationary F0 subunits as thec 12-γε subunits rotate as a unit. The γ and ε subunits are known to project below the α3β3 complex as a stalk making contact with the surface of the c-oligomer in F0 (21Zhang Y. Fillingame R.H. J. Biol. Chem. 1995; 270: 24609-24614Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 22Watts S.D. Zhang Y. Fillingame R.H. Capaldi R.A. FEBS Lett. 1995; 368: 235-238Crossref PubMed Scopus (84) Google Scholar, 23Watts S.D. Teng C. Capaldi R.A. J. Biol. Chem. 1996; 271: 28341-28347Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Recent electron micrographs now indicate a second stalk at the periphery of the F1F0 interface, which is presumed to represent a dimer of b 2 subunits extending from F0 to F1 (7Walker J.E. Angew. Chem. Int. Ed. Engl. 1998; 37: 2308-2319Crossref PubMed Scopus (248) Google Scholar, 24Wilkens S. Capaldi R.A. Nature. 1998; 393: 29Crossref PubMed Scopus (135) Google Scholar, 25Böttcher B. Schwarz L. Gräber P. J. Mol. Biol. 1998; 281: 757-762Crossref PubMed Scopus (87) Google Scholar). Little is known about the structure of subunit b. Subunit b is an amphipathic protein of 156 residues. The N-terminal 33-residue segment is highly hydrophobic and the presumed membrane anchor (26Walker J.E. Saraste M. Gay J.E. Biochim. Biophys. Acta. 1984; 768: 164-200Crossref PubMed Scopus (371) Google Scholar). Indeed, residues in this N-terminal segment were readily labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a lipid soluble, photoactivatable carbene precursor (27Hoppe J. Brunner J. Jørgensen B.B. Biochemistry. 1984; 23: 5610-5616Crossref PubMed Scopus (70) Google Scholar). The remainder of the protein is quite hydrophilic and thought to extend from the membrane surface to bind F1. The cytoplasmic domain lacking residues 1–24, termed b sol, has been expressed and in purified, soluble form shown to bind to F1 (28Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar). The cytoplasmic domain has an elongated shape with high α-helical content and associates to form a homodimer in solution. Dimerization appears to be a necessary prerequisite for F1 binding (29Rodgers A.J.W. Wilkens S. Aggeler R. Morris M.B. Howitt S.M. Capaldi R.A. J. Biol. Chem. 1997; 272: 31058-31064Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 30Sorgen P.L. Bubb M.R. McCormick K.A. Edison A.S. Cain B.D. Biochemistry. 1998; 37: 923-932Crossref PubMed Scopus (47) Google Scholar). The soluble domain binds to subunit δ of F1 in solution (31Dunn S.D. Chandler J. J. Biol. Chem. 1998; 273: 8646-8651Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and interactions between subunit b and subunits δ and α at the top of the F1 molecule have been demonstrated in F1F0 (32Rodgers A.J.W. Capaldi R.A. J. Biol. Chem. 1998; 273: 29406-29410Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). To reach the top of the F1 molecule, subunit b is estimated to extend 110 Å from the surface of the membrane (32Rodgers A.J.W. Capaldi R.A. J. Biol. Chem. 1998; 273: 29406-29410Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). It may be possible to solve the structure of the individual domains of subunit b by NMR methods as a means of circumventing solubility problems inherent in approaches with detergent solubilization of the whole subunit. The aqueous solution structures of subunit ε and portions of subunit δ are already in hand (33Wilkens S. Dahlquist F.W. McIntosh L.P. Donaldson L.W. Capaldi R.A. Nat. Struct. Biol. 1995; 2: 961-967Crossref PubMed Scopus (157) Google Scholar, 34Wilkens S. Dunn S.D. Chandler J. Dahlquist F.W. Capaldi R.A. Nat. Struct. Biol. 1995; 4: 198-201Crossref Scopus (110) Google Scholar), and the structure of subunit c in chloroform/methanol/H2O solvent agrees well with predictions made from the biochemical and genetic experiments on the protein in situ (2Fillingame R.H. J. Exp. Biol. 1997; 200: 217-224Crossref PubMed Google Scholar, 11Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 37: 8817-8824Crossref PubMed Scopus (272) Google Scholar, 13Fillingame R.H. Jones P.C. Jiang W. Valiyaveetil F.I. Dmitriev O.Y. Biochim. Biophys. Acta. 1998; 1365: 135-142Crossref PubMed Scopus (59) Google Scholar). Further, subunit cretains its function after passage through chloroform/methanol/H2O solvent when reconstituted into liposomes with subunits a and b (35Dmitriev O.Y. Altendorf K. Fillingame R.H. Eur. J. Biochem. 1995; 223: 478-483Crossref Scopus (45) Google Scholar). Based on its hydrophobicity, the membrane anchoring segment of subunitb was expected to be soluble in the chloroform/methanol/H2O mixture used in studies of subunitc. We report here on the structure of the membrane anchoring segment of subunit b in chloroform/methanol/H2O solvent and its possible relevance to the structural organization of the native subunit b dimer in F0. A 34-residue peptide corresponding to the N-terminal sequence of the E. coli subunit b was synthesized at the University of Wisconsin Biotechnology Center on an Applied Biosystems Synergy 432A instrument using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. The C-terminal carboxyl was amidinated. The peptide was purified from the crude synthesis mixture by reverse phase high pressure liquid chromatography on a Dynamax C-4 column eluted with a linear 55–72% gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid. The identity of the purified peptide was confirmed by amino acid analysis and electrospray mass spectrometry. The final product was judged to be ≥99% pure based on analytical high pressure liquid chromatography. Samples for NMR were 2 mmpeptide in either CDCl3:CD3OH:H2O (4:4:1 by volume) or CDCl3:CD3OD:D2O (4:4:1 by volume) containing 50 mm NaCl and 1 mm dithiothreitol. The pH of the solution was measured with a glass electrode and adjusted to pH 6.0 without correction for the deuterium isotope effect. DQF-COSY, 1The abbreviations used are: DQF-COSY, double-quantum filtered correlation spectroscopy; NOE, nuclear Overhauser enhancement; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; PCR, polymerase chain reaction TOCSY, and NOESY experiments were performed on a DMX-600 spectrometer (Brueker) with a triple axis gradient capability. Double pulse field gradient echo solvent suppression (36Hwang T.L. Shaka A.J. J. Magn. Reson. (A). 1995; 112: 275-279Crossref Scopus (1565) Google Scholar) was used for recording TOCSY and NOESY in protic solvent. Magic angle gradients (37Warren W.S. Richter W. Andreotti A.H. Farmer B.T. Science. 1993; 262: 2005-2009Crossref PubMed Scopus (354) Google Scholar) were used for coherence pathway selection and water suppression in DQF-COSY experiments. TOCSY used the DIPSI-2 spin-lock sequence (38Shaka A.J. Lee C.J. Pines A. J. Magn. Reson. 1988; 77: 274-293Google Scholar) and a mixing time of 75 ms. A mixing time of 160 or 80 ms was used in NOESY experiments. Data was collected using 640 (NOESY, TOCSY) or 800 (DQF-COSY) increments int 1. Time domain data was also extended int 1 by linear prediction. Squared sine apodization and zero filling to 2,048 points was applied in each dimension before Fourier transformation. Spectra were processed and analyzed using Felix 95.0 software (Molecular Simulations Inc., Palo Alto, CA) on a Silicon Graphics O2 computer. Coupling constants (3 J Hα, HN) were calculated from DQF-COSY and NOESY spectra essentially as described (39Titman J.J. Keeler J. J. Magn. Reson. 1990; 89: 640-646Google Scholar). The structure was calculated from 275 NOE-derived inter- and intra-residue distance constraints and 20 angle constraints derived from coupling constants. Distance calibration and structure calculation was performed with the DYANA software package (40Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar) by simulated annealing. The MOLMOL program (41Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6490) Google Scholar) was used for visual analysis of the structure and for preparing molecular graphics figures. A two stage PCR-based mutagenesis procedure was used with plasmid pNOC for the introduction of single cysteine residues between residues 2 and 20 of subunit b (12Jones P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1998; 273: 17178-17185Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Mutagenic primers corresponding to 21–25 base sequence of the sense strand were designed with a single codon changed to Cys. These were combined with the antisense primer (bases 2523–2548) 2The nucleotide numbering system is from the sequence given by Walker et al. (26Walker J.E. Saraste M. Gay J.E. Biochim. Biophys. Acta. 1984; 768: 164-200Crossref PubMed Scopus (371) Google Scholar). to generate the first PCR product or mega-primer. The purified mega-primer was then combined with a second primer, coding bases 1844–1860 of the sense strand, in a second PCR reaction. The product was then digested withSnaB1 and AvaI and subcloned into these restriction sites of plasmid pNOC, and the product was verified by DNA sequencing. A chromosomal ΔuncBEFH deleted strain, JWP109 (17Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar), was transformed with pNOC and its mutant derivatives. Plasmid complementation of the ΔuncBEFH deletion was tested by growth of transformant cells on succinate minimal medium (17Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar). Membrane vesicles were prepared, and cross-linking analysis was carried out as described previously (17Jiang W. Fillingame R.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6607-6612Crossref PubMed Scopus (150) Google Scholar) using polyvinylidene fluoride membrane for Western blotting. Rabbit antiserum to subunit b was a generous gift of D. S. Perlin and A. E. Senior (42Perlin D.S. Senior A.E. Arch. Biochem. Biophys. 1985; 236: 603-611Crossref PubMed Scopus (31) Google Scholar). Peptideb 1–34 is quite hydrophobic and it proved to be very soluble in a mixture of chloroform/methanol/H2O (4:4:1 by volume). The DQF-COSY spectrum of b 1–34 in this solvent mixture demonstrated a good dispersion of HNand Hα chemical shifts (Fig.1). The distribution of chemical shifts of the HN and Hα protons is typical for a protein containing α-helical and coiled segments (43Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-391Crossref PubMed Scopus (937) Google Scholar). Proton chemical shifts were assigned by standard procedures (44Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York1986Crossref Google Scholar). Main chain chemical shifts assignments for all residues were complete with the exception of Asn-2, where HN was not observed. Most of the side chains protons have been assigned except for a few aliphatic side chains where complete assignment was not possible due to spectral overlap and chemical shift degeneracy. A table of chemical shift assignments has been deposited in the BMRB data bank (http://www.bmrb.wisc.edu). NOE analysis revealed a pattern of sequential and medium range NOEs, which is characteristic of an α-helix (Fig.2 A). No long range NOEs were observed (Table I), indicating that the peptide does not form tertiary folds. The Hα-HN cross-peaks of the residues 12–21 and 25 were still readily observable by DQF-COSY in completely deuterated solvent in an experiment where data was collected from 6 to 14 h after dissolving the peptide. These regions of the peptide must therefore have a particularly stable hydrogen bonded secondary structure.Figure 2Summary of NOEs observed in the NOESY spectrum of the b 1–34 peptide. A, sequential and medium range NOEs and slowly exchanging amide protons; B, number of NOE constraints per residue. Segments of the bars correspond to interresidue (white), sequential (light shading) and other (dark shading) NOEs, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IStatistics for 10 final structural models from DYANA calculationsNOE-derived distance constraints (total = 275)Intraresidue92Sequential (i = 1)89Short range (1 < ‖i − j‖ ≤ 4)94Long range (‖i − j‖ > 4)0Coupling constant derived ihedral angle constraints (total = 20)φ19χ11Mean global pairwise backbone RMSD0.37 ± 0.14 ÅMean global pairwise all heavy atom RMSD0.89 ± 0.14 ÅNumber of consistent constraint violationsDistance violations ≥0.2 Å0Angle violations ≥5°1 Open table in a new tab The three-dimensional structure ofb 1–34 was calculated by simulated annealing with the DYANA package of NMR software (40Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar). Of the initial collection of 200 calculated structures, 180 had a similar overall folding pattern. In the 20 remaining structures there was no apparent clustering of structures into an alternative fold. The 10 lowest energy structures were energy minimized using AMBER forcefield as implemented in DISCOVER (Molecular Simulations Inc.). The atomic coordinates of the 10 final structures have been deposited as entry 1b9U at the Protein Data Bank, Rutgers, New Jersey. The best fit superposition of the 10 final structures is shown in Fig. 3. Mean pairwise root mean square deviation between individual structures for residues 3–33 was 0.4 ± 0.1 Å and 0.9 ± 0.1 Å for the backbone and all heavy atoms, respectively. There were no distance constraint violations exceeding 0.2 Å. The distribution of angles in a Ramachandran plot were 74% in the “most favored” region, with 23% in the “additionally allowed” and 3% in “generously allowed” regions. The definitions of the Ramachandran plot regions are those used in DYANA. Statistics on the structure calculation are presented in Table I and Fig. 2 B. The b 1–34 peptide forms a well defined α-helix from residues 4–22, which is interrupted by a bend region from residues 23–26 with resumption of the α-helix from residue 27 to the C terminus. A large stretch of the initial α-helical segment is unusually stable as judged by the very slow HN exchange in deuterated solvent. The uninterrupted stretch of hydrophobic side chains from residues 11 to 20 may stabilize the hydrogen bonded secondary structure in either a lipid bilayer or a membrane-mimetic solvent by forming a nonpolar sheath around the protein backbone. Sequential proline residues at positions 27 and 28, which would break the (i,i + 4) pattern of hydrogen bonding in an α-helix, correlate with the position of the bend in the structure. This region is well ordered in solution despite the absence of hydrogen bonds. Such a rigid conformation probably results from the combination of restricted torsional mobility of the backbone of the two proline residues and spatial constraints imposed by the bulky side chains of residues 22–26. The α-helical structure resumes at residue 27 with both Pro-27 and Pro-28 in the α-helical conformation with ψ angles of about −35°. As Richardson and Richardson (45Richardson J.S. Richardson D.C. Fasman G. Prediction of Protein Structure and the Principles of Protein Conformation. Plenum Press, NY1989: 1-98Crossref Google Scholar) have indicated, a proline in this conformation is actually favored as the first residue of an α-helix and is not uncommon in the second position as well. The α-helix from residues 27 to 33 may be part of a more extended α-helical segment of the cytoplasmic domain. The cytoplasmic domains of subunit b forms a functionally important homodimer in F0, which suggests a possible proximity of transmembrane segments as well. Single Cys residues were introduced from position 2–20 of a cysteine-free subunitb (bC21S) to test this possibility by cross-linking. The bC21S mutant was shown to be functionally equivalent to wild type (12Jones P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1998; 273: 17178-17185Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Each of the single Cys mutants grew on a succinate carbon source, indicating a functional oxidative phosphorylation system. Most mutants grew similarly to wild type (1.5–2-mm colony diameter after 3 days at 37 °C) with the exception of bI7C (0.5 mm), bG9C (0.8 mm), andbI12C (1 mm). Membrane vesicles from each mutant were analyzed for b-b homodimer formation by disulfide bridge formation following treatment with Cu(II)-(1,10-phenanthroline)2 (Fig.4). Relatively intense high yieldb-b dimers were observed for the bN2C,bT6C and bQ10C substitutions. Less intense dimer formation was observed with Cys substitutions at residues 3, 4, 8, 9, 11, 13, 14, 17, and 18. The periodicity of high yield cross-linking seen in Fig. 4 mimics that expected for one face of an α-helix. The less intense cross-linking seen over consecutive stretches of residues suggest that the helices may be relatively mobile in the membrane. Intersubunit distance restraints derived from the cross-linking pattern were used to envision the orientation of the membrane domains of the two b subunits in the native F0 complex. Two minimized meanb 1–34 structures were docked to each other using distance constraints from the cross-linking data. The distances between the α-carbons of residues 2, 6, and 10 of two differentb subunits were constrained to 4–8 Å, the distance usually found for natural disulfide bridges in proteins (45Richardson J.S. Richardson D.C. Fasman G. Prediction of Protein Structure and the Principles of Protein Conformation. Plenum Press, NY1989: 1-98Crossref Google Scholar). The distances between αcarbons of residues forming lower yield cross-links were constrained to 4–11 Å. Backbone angles of residues 3 to 33 were restrained to the values in the mean structure, and side chain angles left unrestrained. A dimer structure was calculated using molecular dynamics and energy minimization with DISCOVER (Molecular Simulations Inc.). The fit of the cross-linking distance constraints to the model are shown in Table II. The modeling indicates that the membrane anchoring segments of the two bsubunits are positioned at a 23° angle to each other with interacting helical faces making Van der Waals contact at the side chains of residues 6 and 10 (Fig. 5).Table IICross-linking distance constraints used in modeling the b1–34dimerResidue pairaPrime designates residue of second subunit in dimer.Distance contraint rangeFinal distanceÅ2–2′4–88.13–3′4–114.54–4′4–1110.96–6′4–85.28–8′4–1111.69–9′4–1110.610–10′4–85.211–11′4–1110.513–13′4–1110.414–14′4–117.617–17′4–1110.318–18′4–1111.1a Prime designates residue of second subunit in dimer. Open table in a new tab Before this work, Girvin et al. (11Girvin M.E. Rastogi V.K. Abildgaard F. Markley J.L. Fillingame R.H. Biochemistry. 1998; 37: 8817-8824Crossref PubMed Scopus (272) Google Scholar) used solution NMR to solve the structure of F0 subunit c dissolved in chloroform/methanol/H2O (4:4:1) solvent. Importantly, subunit c folds in a helical hairpin, as it is predicted to fold in the membrane, with a number of side chains interacting in accord with the predictions of genetic and biochemical studies of F0 in situ. The solvent mixture used may be a good membrane mimetic, because it can organize heterogeneously around polar and apolar surfaces of amphipathic proteins. We have shown previously that purified subunit c, prepared in chloroform/methanol/H2O solvent, can be reconstituted with subunits a and b to form an F0 with normal proton translocating function (35Dmitriev O.Y. Altendorf K. Fillingame R.H. Eur. J. Biochem. 1995; 223: 478-483Crossref Scopus (45) Google Scholar). The experiment indicated that the protein was not denatured by the solvent treatment. We have attempted similar experiments here in reconstituting peptideb 1–34 with purified subunits a andc and were unable to reconstitute proton-translocating activity. This negative result is in agreement with earlier observations that partial removal of small segments of the C-terminal domain of the subunit b disrupted the assembly of an active F0 complex (46Steffens K. Schneider E. Deckers-Hebestreit G. Altendorf K. J. Biol. Chem. 1986; 262: 5866-5869Abstract Full Text PDF Google Scholar, 47Takeyama M. Noumi T. Maeda M. Futai M. J. Biol. Chem. 1988; 263: 16106-16112Abstract Full Text PDF PubMed Google Scholar). The structure of the transmembrane region of subunit bderived here by NMR analysis of the protein in chloroform/methanol/H2O solvent fits well with features of the protein expected in a native lipid bilayer. The N-terminal α-helical segment (residues 4–22) is followed by hinge region from residues 23–26 before resumption of the α-helix. Because an α-helix of 20 amino acids is required to traverse the fatty acyl hydrocarbon interior of a phospholipid bilayer, we expect the initial N-terminal helix to be the hydrocarbon spanning region of the protein. This would place Asn-2 at the periplasmic hydrocarbon/polar interface and the hinge region (residues 23–26) at the cytoplasmic hydrocarbon/polar interface of the phospholipid bilayer. The distance between α-carbons of Asn-2 and Trp-26 is 34 Å in the structure, which is close to the distance of 32 Å predicted between fatty acyl carbonyls in opposing leaflets of a palmitoyloleoylphosphatidylcholine bilayer determined by x-ray and neutron diffraction (48Wiener M.C. White S. Biophys. J. 1992; 61: 434-447Abstract Full Text PDF PubMed Scopus (652) Google Scholar, 49Yau W.-M. Wimley W.C. Gawrisch K. White S.H. Biochemistry. 1998; 37: 14713-14718Crossref PubMed Scopus (831) Google Scholar). The positioning of these residues near the glycerol moiety of the phospholipid was previously indicated by labeling studies with a photoactivatable phospholipid analog (50Hoppe J. Montecucco C. Friedl P. J. Biol. Chem. 1983; 258: 2882-2885Abstract Full Text PDF PubMed Google Scholar). Trp and Tyr residues are preferentially found at the hydrocarbon/polar interface of the lipid bilayer in transmembrane proteins of known structure (49Yau W.-M. Wimley W.C. Gawrisch K. White S.H. Biochemistry. 1998; 37: 14713-14718Crossref PubMed Scopus (831) Google Scholar, 51von Heijne G. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 167-192Crossref PubMed Scopus (255) Google Scholar, 52Reithmeier R.A.F. Curr. Opin. Struct. Biol. 1995; 5: 491-500Crossref PubMed Scopus (143) Google Scholar, 53Wallin E. Tsukihara T. Yoshikawa S. von Heijne G. Elofsson A. Protein Sci. 1995; 6: 808-815Crossref Scopus (132) Google Scholar). The Tyr-24 and Trp-26 residues in theb 1–34 structure would also be predicted to organize in the interfacial region. In the case of the Trp-26 side chain, the aromatic rings lie parallel to the predicted surface of the lipid bilayer but in a region of the protein devoid of other protein contacts (Fig. 3). Trp-26 can be replaced with either an acidic or a basic residue without impairing function (54Jans D.A. Fimmel A.L. Hatch L. Gibson F. Cox G.B. J. Bacteriol. 1984; 160: 764-770Crossref PubMed Google Scholar), indicating that it may lie in a region with a few protein-protein contacts. The orientation of the indole ring might be expected to reorient in a phospholipid bilayer in a more perpendicular manner to optimize hydrogen bonding between the indole NH and fatty acyl carbonyl groups (55Schiffer M. Chang C.-H. Stevens F.J. Protein Eng. 1992; 5: 213-214Crossref PubMed Scopus (347) Google Scholar). 3Such a reorientation does occur in the molecular dynamics calculation illustrated in Fig. 5, where side chain angles were left unrestrained. The two copies of subunit b present in the F0are now thought to be adjacent to each other and the interactions between cytoplasmic domains believed critical in F1 binding function (28Dunn S.D. J. Biol. Chem. 1992; 267: 7630-7636Abstract Full Text PDF PubMed Google Scholar, 29Rodgers A.J.W. Wilkens S. Aggeler R. Morris M.B. Howitt S.M. Capaldi R.A. J. Biol. Chem. 1997; 272: 31058-31064Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 30Sorgen P.L. Bubb M.R. McCormick K.A. Edison A.S. Cain B.D. Biochemistry. 1998; 37: 923-932Crossref PubMed Scopus (47) Google Scholar, 31Dunn S.D. Chandler J. J. Biol. Chem. 1998; 273: 8646-8651Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 32Rodgers A.J.W. Capaldi R.A. J. Biol. Chem. 1998; 273: 29406-29410Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The cross-linking experiments presented here indicate that the transmembrane domains are also close enough in the membrane to dimerize. A possible model for helical-helical interaction within the membrane is presented based upon the NMR model and distance constraints from the cross-linking results (Fig. 5). The model is of interest in that the transmembrane helices cross at an angle that is typical for helix-helix packing in polytopic membrane proteins of known structure (56Bowie J.U. J. Mol. Biol. 1997; 272: 780-789Crossref PubMed Scopus (280) Google Scholar). If this model represents the packing in F0, then the role of the hinge region may be to redirect the C-terminal helix at an angle more perpendicular to the membrane as it emerges into the cytoplasmic. Proper positioning may be critical in facilitating dimerization of the cytoplasmic domain. The model is also of interest in that it suggests a possible aromatic cluster that may be important in fostering helix-helix interactions between subunits. The aromatic ring interactions of Phe-14-Phe-17′, where 17′ designates the second subunit, and Phe-17′-Phe-17 is in accord with the stabilizing geometries and distances described by Burley and Petsko (57Burley S.K. Petsko G.A. Science. 1985; 229: 23-28Crossref PubMed Scopus (2242) Google Scholar) with centroid distances of 5.0 and 5.8 Å, respectively. We are grateful to Dr. Gary Case for synthesizing the peptide used in this study and to Dr. Mark Girvin for introducing O. Dmitriev to NMR. We thank Drs. David Perlin (Public Health Institute of New York) and Alan Senior (University of Rochester) for the gift of antiserum to subunit b." @default.
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• W2010071674 title "Structure of the Membrane Domain of Subunit b of theEscherichia coli F0F1 ATP Synthase" @default.
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