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• W2144937827 abstract "The rotation of F1Fo-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane Fo domain, which is coupled to the ATP-producing F1 domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F1Fo-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode. The rotation of F1Fo-ATP synthase is powered by the proton motive force across the energy-transducing membrane. The protein complex functions like a turbine; the proton flow drives the rotation of the c-ring of the transmembrane Fo domain, which is coupled to the ATP-producing F1 domain. The hairpin-structured c-protomers transport the protons by reversible protonation/deprotonation of a conserved Asp/Glu at the outer transmembrane helix (TMH). An open question is the proton transfer pathway through the membrane at atomic resolution. The protons are thought to be transferred via two half-channels to and from the conserved cAsp/Glu in the middle of the membrane. By molecular dynamics simulations of c-ring structures in a lipid bilayer, we mapped a water channel as one of the half-channels. We also analyzed the suppressor mutant cP24D/E61G in which the functional carboxylate is shifted to the inner TMH of the c-protomers. Current models concentrating on the “locked” and “open” conformations of the conserved carboxylate side chain are unable to explain the molecular function of this mutant. Our molecular dynamics simulations revealed an extended water channel with additional water molecules bridging the distance of the outer to the inner TMH. We suggest that the geometry of the water channel is an important feature for the molecular function of the membrane part of F1Fo-ATP synthase. The inclination of the proton pathway isolates the two half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode. ATP synthases from bacteria, mitochondria, and chloroplasts produce ATP, the universal fuel in biological cells. To synthesize the high energy triphosphate from ADP and inorganic phosphate, the enzymes use a transmembrane proton gradient (1Stock D. Leslie A.G. Walker J.E. Molecular architecture of the rotary motor in ATP synthase.Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1085) Google Scholar, 2von Ballmoos C. Wiedenmann A. Dimroth P. Essentials for ATP synthesis by F1F0 ATP synthases.Annu. Rev. Biochem. 2009; 78: 649-672Crossref PubMed Scopus (264) Google Scholar, 3Junge W. Sielaff H. Engelbrecht S. Torque generation and elastic power transmission in the rotary FOF1-ATPase.Nature. 2009; 459: 364-370Crossref PubMed Scopus (295) Google Scholar, 4Vik S.B. Antonio B.J. A mechanism of proton translocation by F1F0 ATP synthases suggested by double mutants of the a subunit.J. Biol. Chem. 1994; 269: 30364-30369Abstract Full Text PDF PubMed Google Scholar) and convert electrochemical into chemical energy. F-type ATP synthases consist of a transmembrane Fo domain and an extramembranous F1 domain. Fo from chloroplasts comprises a rotating c-ring and a stator domain formed by subunits a, b, and b′. Although the stoichiometry of the transmembrane stator domain is fixed in all organisms, the c-ring has 8 subunits in bovine mitochondria (5Watt I.N. Montgomery M.G. Runswick M.J. Leslie A.G. Walker J.E. Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 16823-16827Crossref PubMed Scopus (368) Google Scholar), 10 subunits in yeast mitochondria (1Stock D. Leslie A.G. Walker J.E. Molecular architecture of the rotary motor in ATP synthase.Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1085) Google Scholar), 14 subunits in chloroplasts (6Seelert H. Poetsch A. Dencher N.A. Engel A. Stahlberg H. Müller D.J. Proton-powered turbine of a plant motor.Nature. 2000; 405: 418-419Crossref PubMed Scopus (414) Google Scholar, 7Vollmar M. Schlieper D. Winn M. Büchner C. Groth G. Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase.J. Biol. Chem. 2009; 284: 18228-18235Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and 10–15 subunits in bacteria (8Jiang W. Hermolin J. Fillingame R.H. The preferred stoichiometry of c subunits in the rotary motor sector of Escherichia coli ATP synthase is 10.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 4966-4971Crossref PubMed Scopus (215) Google Scholar, 9Mitome N. Suzuki T. Hayashi S. Yoshida M. Thermophilic ATP synthase has a decamer c-ring: indication of noninteger 10:3 H+/ATP ratio and permissive elastic coupling.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12159-12164Crossref PubMed Scopus (106) Google Scholar, 10Stahlberg H. Müller D.J. Suda K. Fotiadis D. Engel A. Meier T. Matthey U. Dimroth P. Bacterial Na+-ATP synthase has an undecameric rotor.EMBO Rep. 2001; 2: 229-233Crossref PubMed Scopus (168) Google Scholar, 11Meier T. Matthey U. von Ballmoos C. Vonck J. Krug von Nidda T. Kühlbrandt W. Dimroth P. Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases.J. Mol. Biol. 2003; 325: 389-397Crossref PubMed Scopus (75) Google Scholar, 12Pogoryelov D. Yu J. Meier T. Vonck J. Dimroth P. Muller D.J. The c15-ring of the Spirulina platensis F-ATP synthase: F1/F0 symmetry mismatch is not obligatory.EMBO Rep. 2005; 6: 1040-1044Crossref PubMed Scopus (140) Google Scholar, 13Meier T. Ferguson S.A. Cook G.M. Dimroth P. Vonck J. Structural investigations of the membrane-embedded rotor ring of the F-ATPase from Clostridium paradoxum.J. Bacteriol. 2006; 188: 7759-7764Crossref PubMed Scopus (52) Google Scholar, 14Meier T. Morgner N. Matthies D. Pogoryelov D. Keis S. Cook G.M. Dimroth P. Brutschy B. A tridecameric c-ring of the adenosine triphosphate (ATP) synthase from the thermoalkaliphilic Bacillus sp. strain TA2.A1 facilitates ATP synthesis at low electrochemical proton potential.Mol. Microbiol. 2007; 65: 1181-1192Crossref PubMed Scopus (76) Google Scholar, 15Pogoryelov D. Reichen C. Klyszejko A.L. Brunisholz R. Muller D.J. Dimroth P. Meier T. The oligomeric state of c-rings from cyanobacterial F-ATP synthases varies from 13 to 15.J. Bacteriol. 2007; 189: 5895-5902Crossref PubMed Scopus (81) Google Scholar). The chloroplast F1 domain comprises subunits γ and ϵ, which rotate with the c-ring, and the nonrotating catalytic domain α3β3, which is connected to the stator domain of Fo through the δ subunit (see Fig. 1A). Light-driven proton pumps in chloroplasts and phototropic bacteria or respiratory-chain enzymes in mitochondria and aerobic bacteria generate a proton gradient across the membrane, resulting in a positively charged p-side 3The abbreviations used are: p-sidepositive side of the membranen-sidenegative side of the membraneMDmolecular dynamicsNPATconstant area isobaric-isothermal ensembleNPTisobaric-isothermal ensembleNVTcanonical ensembler.m.s.d.root mean square deviation(s)TMHtransmembrane helixPDBProtein Data Bank. and a negatively charged n-side (p-side and n-side refer to lumen and stroma in chloroplasts, intermembrane space and matrix in mitochondria, and periplasm and cytoplasm in bacteria, respectively). The resulting proton motive force drives the c8–15γϵ rotor, which induces conformational changes in the β subunits of the stator and, hence, provides the energy to produce ATP. Some fermenting bacteria use sodium ions instead of protons to drive ATP synthesis (16Dimroth P. Wang H. Grabe M. Oster G. Energy transduction in the sodium F-ATPase of Propionigenium modestum.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 4924-4929Crossref PubMed Scopus (134) Google Scholar). positive side of the membrane negative side of the membrane molecular dynamics constant area isobaric-isothermal ensemble isobaric-isothermal ensemble canonical ensemble root mean square deviation(s) transmembrane helix Protein Data Bank. Subunits a and c of the transmembrane Fo domain are directly involved in proton transport. Transport from the p-side to the middle of the membrane where protons bind to a conserved carboxylate of subunit c (Glu61 in chloroplasts) is facilitated by an intrinsic channel or proton wire in subunit a (p-side half-channel) (17Vik S.B. Patterson A.R. Antonio B.J. Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel.J. Biol. Chem. 1998; 273 (Correction (1998) J. Biol. Chem.273, 22159): 16229-16234Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 18Angevine C.M. Fillingame R.H. Aqueous access channels in subunit a of rotary ATP synthase.J. Biol. Chem. 2003; 278: 6066-6074Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Angevine C.M. Herold K.A. Fillingame R.H. Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13179-13183Crossref PubMed Scopus (70) Google Scholar, 20Schwem B.E. Fillingame R.H. Cross-linking between helices within subunit a of Escherichia coli ATP synthase defines the transmembrane packing of a four-helix bundle.J. Biol. Chem. 2006; 281: 37861-37867Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 21Angevine C.M. Herold K.A. Vincent O.D. Fillingame R.H. Aqueous access pathway in ATP synthase subunit a: reactivity of cysteine substituted into transmembrane helices 1, 3, and 5.J. Biol. Chem. 2007; 282: 9001-9007Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 22Dong H. Fillingame R.H. Chemical reactivities of cysteine substitutions in subunit a of ATP synthase define residues gating H+ transport from each side of the membrane.J. Biol. Chem. 2010; 285: 39811-39818Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Protonation of the essential carboxylate in the c-subunit drives clockwise rotation of the entire c-ring (as seen from the p-side) (23Börsch M. Diez M. Zimmermann B. Reuter R. Gräber P. Stepwise rotation of the γ-subunit of EF0F1-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer.FEBS Lett. 2002; 527: 147-152Crossref PubMed Scopus (114) Google Scholar). The step size of the c-ring corresponds to one subunit of c, indicating a proton motive force-driven Brownian ratchet mechanism (24Düser M.G. Zarrabi N. Cipriano D.J. Ernst S. Glick G.D. Dunn S.D. Börsch M. 36° step size of proton-driven c-ring rotation in FoF1-ATP synthase.EMBO J. 2009; 28: 2689-2696Crossref PubMed Scopus (98) Google Scholar, 25Ishmukhametov R. Hornung T. Spetzler D. Frasch W.D. Direct observation of stepped proteolipid ring rotation in E. coli FoF1-ATP synthase.EMBO J. 2010; 29: 3911-3923Crossref PubMed Scopus (82) Google Scholar). The protons are then released from the proton-binding site on subunit c to the n-side (17Vik S.B. Patterson A.R. Antonio B.J. Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel.J. Biol. Chem. 1998; 273 (Correction (1998) J. Biol. Chem.273, 22159): 16229-16234Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26Junge W. Lill H. Engelbrecht S. ATP synthase: an electrochemical transducer with rotatory mechanics.Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (437) Google Scholar). An aqueous path at the interface between subunits a and c was inferred by mutagenesis and labeling studies (18Angevine C.M. Fillingame R.H. Aqueous access channels in subunit a of rotary ATP synthase.J. Biol. Chem. 2003; 278: 6066-6074Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Angevine C.M. Herold K.A. Fillingame R.H. Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13179-13183Crossref PubMed Scopus (70) Google Scholar, 21Angevine C.M. Herold K.A. Vincent O.D. Fillingame R.H. Aqueous access pathway in ATP synthase subunit a: reactivity of cysteine substituted into transmembrane helices 1, 3, and 5.J. Biol. Chem. 2007; 282: 9001-9007Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 27Moore K.J. Angevine C.M. Vincent O.D. Schwem B.E. Fillingame R.H. The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism.J. Biol. Chem. 2008; 283: 13044-13052Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 28Steed P.R. Fillingame R.H. Subunit a facilitates aqueous access to a membrane-embedded region of subunit c in Escherichia coli F1F0 ATP synthase.J. Biol. Chem. 2008; 283: 12365-12372Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 29Steed P.R. Fillingame R.H. Aqueous accessibility to the transmembrane regions of subunit c of the Escherichia coli F1F0 ATP synthase.J. Biol. Chem. 2009; 284: 23243-23250Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), which may function as the n-side half of the proton channel. F1Fo-ATP synthases can also catalyze the reverse reaction driven by ATP hydrolysis and pump protons from the n-side to the p-side of the membrane via the same half-channels, resulting in a counterclockwise rotation of the c-ring rotor (23Börsch M. Diez M. Zimmermann B. Reuter R. Gräber P. Stepwise rotation of the γ-subunit of EF0F1-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer.FEBS Lett. 2002; 527: 147-152Crossref PubMed Scopus (114) Google Scholar). Preventing a nonproductive shortcut between the p-side and the n-side half-channels is an essential prerequisite for the transformation of proton flux into mechanical energy (26Junge W. Lill H. Engelbrecht S. ATP synthase: an electrochemical transducer with rotatory mechanics.Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (437) Google Scholar). A conserved positively charged side chain in subunit a (Arg193 in chloroplasts and Arg210 in Escherichia coli) isolates the two half-channels by preventing any protonated (and thus uncharged) Glu61 of subunit c to pass the positively charged guanidinium group of the aArg193 side chain (30Cain B.D. Simoni R.D. Proton translocation by the F1F0-ATPase of Escherichia coli: mutagenic analysis of the a subunit.J. Biol. Chem. 1989; 264: 3292-3300Abstract Full Text PDF PubMed Google Scholar, 31Hatch L.P. Cox G.B. Howitt S.M. 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. 1995; 270: 29407-29412Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 32Valiyaveetil F.I. Fillingame R.H. On the role of Arg-210 and Glu-219 of subunit a in proton translocation by the Escherichia coli F0F1-ATP synthase.J. Biol. Chem. 1997; 272: 32635-32641Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 33Langemeyer L. Engelbrecht S. Essential arginine in subunit a and aspartate in subunit c of FoF1 ATP synthase: effect of repositioning with helix 4 of subunit a and helix 2 of subunit c.BBA-Bioenergetics. 2007; 1767: 998-1005Crossref Scopus (17) Google Scholar, 34Ishmukhametov R.R. Pond J.B. Al-Huqail A. Galkin M.A. Vik S.B. ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase.Biochim. Biophys. Acta. 2008; 1777: 32-38Crossref PubMed Scopus (25) Google Scholar, 35Mitome N. Ono S. Sato H. Suzuki T. Sone N. Yoshida M. Essential arginine residue of the F0-a subunit in F0F1-ATP synthase has a role to prevent the proton shortcut without c-ring rotation in the F0 proton channel.Biochem. J. 2010; 430: 171-177Crossref PubMed Scopus (56) Google Scholar). Thus, aArg193 prevents the futile counterclockwise rotation in ATP synthesis mode. However, the structure of the a/c interface and how the protein complex maintains isolation between the half-channels, i.e. how the nonrotating shortcut of protons is prevented, has remained elusive so far. Although it is established that the n-side half-channel is water-accessible, the pathway of the protons has not yet been identified. Here we resolve this pathway at atomic resolution by MD simulations of the structure of the c14 rotor ring of the chloroplast ATP synthase (7Vollmar M. Schlieper D. Winn M. Büchner C. Groth G. Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase.J. Biol. Chem. 2009; 284: 18228-18235Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) in a lipid bilayer surrounded by water. The carboxylate cGlu61 of one of the protomers was deprotonated, thus mimicking the protomer facing the a subunit in the intact Fo domain. We identified a water-accessible region inside the lipid bilayer at the periphery of the c-ring rotor. This water channel runs from the n-side of the membrane to the deprotonated carboxylate cGlu61 in the middle of the membrane. The aqueous n-side half-channel is not parallel to the axis of the c-ring rotor but rather inclined, which may enhance the isolation between the half-channels and may contribute to a favorable clockwise rotation in ATP synthesis mode. The MD results also give a plausible explanation of the peculiar properties of the double mutant cP24D/E61G, which, in E. coli, is fully functional despite lacking the essential Asp61 side chain (36Miller M.J. Oldenburg M. Fillingame R.H. The essential carboxyl group in subunit c of the F1F0 ATP synthase can be moved and H+ translocating function retained.Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 4900-4904Crossref PubMed Scopus (126) Google Scholar); the water channel now reaches deep between two c-protomers, thus enabling the repositioning of the reversibly protonated carboxyl group of the suppressor mutant. Starting structures for MD simulations of the c14 rotor ring in a membrane environment were generated from the crystal structure of the c-ring from spinach chloroplasts (Protein Data Bank (PDB) code 2w5j) (7Vollmar M. Schlieper D. Winn M. Büchner C. Groth G. Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase.J. Biol. Chem. 2009; 284: 18228-18235Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). It comprises 14 identical c-subunits, each of which forms a hairpin structure consisting of two TMHs. Missing amino acids and side chains were added manually in the most plausible rotamer conformations and refined as described (7Vollmar M. Schlieper D. Winn M. Büchner C. Groth G. Structure of the c14 rotor ring of the proton translocating chloroplast ATP synthase.J. Biol. Chem. 2009; 284: 18228-18235Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The c-ring structure was oriented with respect to a lipid bilayer according to information provided by the Orientations of Proteins in Membranes database (37Lomize M.A. Lomize A.L. Pogozheva I.D. Mosberg H.I. OPM: orientations of proteins in membranes database.Bioinformatics. 2006; 22: 623-625Crossref PubMed Scopus (892) Google Scholar). The protein structure was then embedded in a bilayer of 288 1,2-dioleoyl-sn-glycero-3-phosphorylcholine lipids, which itself was embedded in 20 Å layers of water molecules above and below. The pre-equilibrated configuration of the lipid bilayer and the water layers was taken from Rosso and Gould (38Rosso L. Gould I.R. Structure and dynamics of phospholipid bilayers using recently developed general all-atom force fields.J. Comput. Chem. 2008; 29: 24-37Crossref PubMed Scopus (72) Google Scholar). Ninety-nine lipids that were sterically overlapping with the protein structure were removed. The center of the c-ring was filled with 12 lipids to model the lipid plug seen in isolated rings (39Meier T. Matthey U. Henzen F. Dimroth P. Müller D.J. The central plug in the reconstituted undecameric c cylinder of a bacterial ATP synthase consists of phospholipids.FEBS Lett. 2001; 505: 353-356Crossref PubMed Scopus (69) Google Scholar). From this system, the following starting structures were generated. I) All but one of the Glu61 residues of the subunits c were protonated (hitherto referred to as “13protonated”). II) All Glu61 residues were protonated (“14protonated”). III) All Glu61 residues were deprotonated (“0protonated”). IV) The double mutant cP24D/E61G was generated by pruning the side chain of Glu61 and replacing Pro24 with the most plausible rotamer of Asp, as provided by Coot (40Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17079) Google Scholar). In this system, all but one of the Asp24 residues were then protonated (double mutant). To reach electroneutrality for each of these starting structures, sodium ions were added by the leap program of the Amber package (41Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. The Amber biomolecular simulation programs.J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6519) Google Scholar) as required. This resulted in system sizes of ∼78,000 atoms. MD simulations were performed with the Amber 10 suite of programs (41Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. The Amber biomolecular simulation programs.J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6519) Google Scholar). For the protein and ions, the force field by Cornell et al. (42Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules.J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11553) Google Scholar) was used with modifications suggested by Simmerling et al. (43Simmerling C. Strockbine B. Roitberg A.E. All-atom structure prediction and folding simulations of a stable protein.J. Am. Chem. Soc. 2002; 124: 11258-11259Crossref PubMed Scopus (540) Google Scholar). TIP3P was used as a water model (44Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. Comparison of simple potential functions for simulating liquid water.J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (29892) Google Scholar). For the lipids, force field parameters and charges derived by Rosso and Gould (38Rosso L. Gould I.R. Structure and dynamics of phospholipid bilayers using recently developed general all-atom force fields.J. Comput. Chem. 2008; 29: 24-37Crossref PubMed Scopus (72) Google Scholar) were used, which are based on the General Amber Force Field (45Wang J. Wolf R.M. Caldwell J.W. Kollman P.A. Case D.A. Development and testing of a general amber force field.J. Comput. Chem. 2004; 25 (Correction (2004) J. Comput. Chem.26, 114): 1157-1174Crossref PubMed Scopus (11698) Google Scholar) and the RESP procedure (46Bayly C.I. Cieplak P. Cornell W.D. Kollman P.A. A well-behaved electrostatic potential based method using charge restraints for deriving atom charges: the RESP model.J. Phys. Chem. 1993; 97: 10269-10280Crossref Scopus (5634) Google Scholar), respectively. These parameters have been shown to yield area per lipid values, peak distances, and lipid volumes that converge around values close to experimental ones when simulations were performed under the condition of an isobaric-isothermal ensemble (NPT) with anisotropic pressure control (38Rosso L. Gould I.R. Structure and dynamics of phospholipid bilayers using recently developed general all-atom force fields.J. Comput. Chem. 2008; 29: 24-37Crossref PubMed Scopus (72) Google Scholar). That way, a restraining of the area as in a constant area isobaric-isothermal (NPAT) ensemble can be avoided, which may lead to simulation artifacts (47Tieleman D.P. Marrink S.J. Berendsen H.J. A computer perspective of membranes: molecular dynamics studies of lipid bilayer systems.Biochim. Biophys. Acta. 1997; 1331: 235-270Crossref PubMed Scopus (692) Google Scholar). Accordingly, after minimization of the systems for 2000 steps with the protein atom positions restrained, canonical ensemble (NVT) MD was carried out for 50 ps, during which the system was heated from 100 to 300 K, applying harmonic restraints with force constants of 5 kcal mol−1 Å−2 to the protein atoms. Subsequent NPT-MD was used for 150 ps to adjust the density. After gradually reducing the force constants of the harmonic restraints on solute atom positions to zero during 250 ps of NPT-MD, the following 20 ns of NPT-MD at 300 K were used to further equilibrate the system. Equilibration times of this length were shown to be necessary for typical simulation studies involving lipid bilayers (48Anézo C. de Vries A.H. Höltje H.-D. Tieleman D.P. Marrink S.-J. Methodological issues in lipid bilayer simulations.J. Phys. Chem. B. 2003; 107: 9424-9433Crossref Scopus (316) Google Scholar). Finally, the following trajectories of 30 ns length generated by NPT-MD were used as production runs with conformations extracted every 20 ps. This resulted in ∼250 ns of total simulation time. Throughout the simulations, the particle mesh Ewald method (49Darden T. York D. Pedersen L. Particle mesh Ewald: an N · log(N) method for Ewald sums in large systems.J. Chem. Phys. 1993; 98: 10089-10092Crossref Scopus (20829) Google Scholar) was used to treat long-range electrostatic interactions, and bond lengths involving bonds to hydrogen atoms were constrained using SHAKE (50Ryckaert J.-P. Ciccotti G. Berendsen H.J. Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes.J. Comput. Phys. 1977; 23: 327-341Crossref Scopus (16906) Google Scholar). The time step for all MD simulations was 2 fs, with a direct space, nonbonded cutoff of 8 Å. The temperature was controlled using the Berendsen thermostat (51Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. DiNola A. Haak J.R. Molecular dynamics with coupling to an external bath.J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23458) Google Scholar) with a time constant of 10 ps, and the Berendsen barostat (51Berendsen H.J.C. Postma J.P.M. van Gunsteren W.F. DiNola A. Haak J.R. Molecular dynamics with coupling to an external bath.J. Chem. Phys. 1984; 81: 3684-3690Crossref Scopus (23458) Google Scholar) was used for anisotropic pressure control with a time constant of 2 ps. The production runs were analyzed with ptraj of the Amber suite of programs (41Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. The Amber biomolecular simulation programs.J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6519) Google Scholar). The “watershell” command was used for analyzing the first and second solvation shells with respect to the oxygen atoms of Glu61 (13protonated, 14protonated, 0protonated) or of Asp24 (double mutant) using lower and upper distances of 3.4 and 5.0 Å, respectively. Water densities were determined with the “grid” command using a cubic grid that encompasses the whole simulation cell with a spacing of 1 Å in each direction. Depicted contour surfaces encompass regions with at least 50 counts of water molecules over the 1,500 snapshots analyzed. Prior to calculating root mean square deviations (r.m.s.d.) and an average structure, the c-ring structure was superimposed with respect to all Cα atoms. A snapshot with a minimal Cα atom r.m.s.d. to the average structure was then taken as the representative structure depicted in FIGURE 1, FIGURE 2. χ1 and χ2 angles of residues Glu61 were determined as the torsion angles involving atoms N-Cα-Cβ-Cγ and Cα-Cβ-Cγ-Cδ, respectively. The kink of the outer helices of the c-ring was determined as the angle of the point triple (center of mass of Cα atoms of residues 48–50; Cα atom of residue 61; center of mass of Cα atoms of residues 74–76). The S.E. was determined as the S.D. divided by the square root of the number of independent snapshots. The number of independent snapshots was determined from the time correlation function for the respective analysis. The correlation time was 200 ps in the case of the watershell analysis and 2.2 ns in the case of the analysis of the χ1 and χ2 torsion angles of Glu61. To analyze possible conformational changes of the c-ring, an elastic network model analysis was performed with the ElNémo webserver (52Suhre K. Sanejouand Y.H. ElNémo: a normal mode web server for protein movement analysis and the generation of templates for molecular replacement.Nucleic Acids Res. 2004; 32: W610-W614Crossref PubMed Scopus (580) Google Scholar) using default parameters. Molecule figures were prepared with PyMOL (Schrödinger, New York, NY). We pe" @default.
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• W2144937827 date "2012-10-01" @default.
• W2144937827 modified "2023-09-30" @default.
• W2144937827 title "Resolving the Negative Potential Side (n-side) Water-accessible Proton Pathway of F-type ATP Synthase by Molecular Dynamics Simulations" @default.
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• W2144937827 doi "https://doi.org/10.1074/jbc.m112.398396" @default.
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