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R H Fillingame

Publications and source records attributed to R H Fillingame.

At least 37 records · Page 2Linked to original sources
PubMed1998

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking.

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1, 10-phenanthroline)2SO4 at 0 degrees, 10 degrees, or 20 degreesC, strong a-c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a-c dimer formation was observed in nine other double mutants after treatment at 20 degreesC in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

Amino Acid Substitution↗
PubMed1997

On the role of Arg-210 and Glu-219 of subunit a in proton translocation by the Escherichia coli F0F1-ATP synthase.

A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the DeltauncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The DeltauncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit a, residues previously postulated to be essential in proton translocation. The aE219G and aE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the aGlu-219 residue cannot play a critical role in proton translocation. The aR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the aR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the DeltapH normally established by NADH oxidation with these membrane vesicles. aR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit a may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.

Arginine↗
PubMed1997

Coupling H+ transport and ATP synthesis in F1F0-ATP synthases: glimpses of interacting parts in a dynamic molecular machine.

Reversible, F1F0-type ATPases (also termed F-ATP synthases) catalyze the synthesis of ATP during oxidative phosphorylation. In animal cells, the enzyme traverses the inner mitochondrial membrane and uses the energy of an H+ electrochemical gradient, generated by electron transport, in coupling H+ translocation to ATP formation. Closely related enzymes are found in the plasma membrane of bacteria such as Escherichia coli, where the enzymes function reversibly depending upon nutritional circumstance. The F1F0-type enzymes are more distantly related to a second family of H(+)-translocating ATPases, the vacuolar-type or V-ATPases. Recent structural information has provided important hints as to how these enzymes couple H+ transport to the chemical work of ATP synthesis. The simplest F1F0-type enzymes, e.g. as in E. coli, are composed of eight types of subunits in an unusual stoichiometry of alpha 3 beta 3 gamma delta epsilon (F1) and a1b2c12 (F0). F1 extends from the membrane, with the alpha and beta subunits alternating around a central subunit gamma. ATP synthesis occurs alternately in different beta subunits, the cooperative tight binding of ADP + Pi at one catalytic site being coupled to ATP release at a second. The differences in binding affinities appear to be caused by rotation of the gamma subunit in the center of the alpha 3 beta 3 hexamer. The gamma subunit traverses a 4.5 nm stalk connecting the catalytic subunits to the membrane-traversing F0 sector. Subunit c is the H(+)-translocating subunit of F0. Protonation/deprotonation of Asp61 in the center of the membrane is coupled to structural changes in an extramembranous loop of subunit c which interacts with both the gamma and epsilon subunits. Subunits gamma and epsilon appear to move from one subunit c to another as ATP is synthesized. The torque of such movement is proposed to cause the rotation of gamma within the alpha 3 beta 3 complex. Four protons are translocated for each ATP synthesized. The movement of gamma and epsilon therefore probably involves a unit of four c subunits. The organization of subunits in F0 remains a mystery; it will have to be understood if we are to understand the mechanism of torque generation.

Adenosine Triphosphatases↗
PubMed1996

Membrane sectors of F- and V-type H+-transporting ATPases.

H+ transporting, reversible F-type ATPases (ATP synthases) and V-type ATPases share major structural features and function by similar mechanisms. Recent structural and genetic experiments provide new insights into the organization of the transmembrane and coupling sectors of the enzymes and the molecular mechanics of coupling H+ transport to ATP.

Amino Acid Sequence↗
PubMed1995

Proton-translocating carboxyl of subunit c of F1Fo H(+)-ATP synthase: the unique environment suggested by the pKa determined by 1H NMR.

Subunit c of the H(+)-transporting F1Fo ATP synthase (EC 3.6.1.34) is thought to fold across the membrane as a hairpin of two alpha helices with a conserved Asp/Glu residue, centered in the second membrane-spanning helix, which is thought to function in H+ translocation. NMR studies indicate that the purified subunit c from Escherichia coli is also folded as a hairpin in a chloroform/methanol/H2O (4:4:1) solvent mixture [Girvin, M. E., & Fillingame, R. H. (1993) Biochemistry 32, 12167-12177] and that the conserved Asp remains uniquely reactive in this solvent mixture [Girvin, M. E., & Fillingame, R. H. (1994) Biochemistry 33, 665-674]. The pKa of Asp61 is of interest because of its unique reactivity and because it is thought to protonate and deprotonate during each proton translocation cycle. We have determined the pKa value of the carboxyl group of the functional Asp in wild type and two functional, mutant subunit c proteins, i.e. the Ala24-->Asp (D24D61) and the Ala24-->Asp/Asp61-->Asn (D24N61) mutant proteins. The pKa values were determined by 1H NMR spectroscopy by measuring changes in the alpha and beta proton chemical shifts by constant time two-dimensional (2D) correlated spectroscopy. The pKa of Asp61 in the purified wild type protein was 7.1. This pKa was significantly higher than the pKa of the other two Asp residues, i.e. Asp7 and Asp44 which were 5.4 and 5.6, respectively. The pKa of the two Glu residues in the protein were determined by 2D total correlation spectroscopy and found to be approximately 5.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Reconstitution of the Fo complex of Escherichia coli ATP synthase from isolated subunits. Varying the number of essential carboxylates by co-incorporation of wild-type and mutant subunit c after purification in organic solvent.

Subunit c of the Escherichia coli F1F0-ATPase, purified in chloroform/methanol (2:1), was reconstituted with detergent-solubilized F0 subunits a and b to form a functionally active H+ channel. The rates of H+ uptake by the proteoliposomes containing the reconstituted F0 complex were comparable to those observed with native F0 reconstituted without subunit dissociation. The F0 reconstituted from purified subunits was also shown to form an active ATP-driven H+ pump upon binding of the F1-ATPase sector of the complex. Reconstitution of D61N and D61G mutant c subunits with wild-type subunits a and b produced an inactive F0. Hybrid F0 complexes, formed with mixtures of wild-type and D61N or D61G mutant c subunits, were also prepared. Formation of an active F0 was prevented by addition of relatively small proportions of D61N or D61G mutant c subunits, i.e. active F0 formation was gradually disrupted as the mutant/wild-type ratio was increased from 0.05 to 0.2. The hybrid reconstitution studies support a model where inactivation of one of the 9-12 c subunits found in F0 is sufficient to abolish activity.

Escherichia coli↗
PubMed1995

Subunits coupling H+ transport and ATP synthesis in the Escherichia coli ATP synthase. Cys-Cys cross-linking of F1 subunit epsilon to the polar loop of F0 subunit c.

Second site suppressor mutations at position 31 of F1 subunit epsilon recouple ATP-driven H+ translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F1F0 ATP synthase (Zhang, Y., Oldenburg, M., and Fillingame, R. H. (1994) J. Biol. Chem. 269, 10221-10224). This finding suggests a functional interaction between subunit c and subunit epsilon during the coupling of H+ transport through F0 to ATP synthesis of F1. However, the physical proximity of the two subunits remained to be defined. In this study, Cys residues were introduced into residues in the polar loop region of subunit c surrounding Gln42 and at position 31 of subunit epsilon to see whether the subunits could be cross-linked. Disulfide bridge formation between subunit c and subunit epsilon was observed in membranes of three double mutants, i.e. cA40C/epsilon E31C, cQ42C/epsilon E31C, and cP43C/epsilon E31C, but not in wild type membranes or in membranes of the cA39C/epsilon E31C double mutant. These results indicate that the polar loop of subunit c and the region around residue 31 of subunit epsilon are physically close to each other in the F1F0 complex and support the hypothesis that these two subunits interact directly in the coupling of H+ transport to ATP synthesis. Disulfide cross-linking of the Q42C subunit c and E31C subunit epsilon leads to inhibition of ATPase coupled H+ transport, as might be expected in a model where the catalytic sites of the F1 ATPase alternate during H+ transport-coupled ATP hydrolysis/synthesis. However, a quantitative relationship between the extent of inhibition of transport and the extent of cross-linking could not be established by the methods used here, and the possibility remains that the epsilon-c cross-linked F1F0 complex retains residual H+ transporting activity.

Adenosine Triphosphate↗
PubMed1995

The gamma subunit in the Escherichia coli ATP synthase complex (ECF1F0) extends through the stalk and contacts the c subunits of the F0 part.

A mutant, in which a cysteine has been site-directed into the polar loop region of the c subunit at residue 44, has been studied. Cross-linking of the c subunit to both the gamma and epsilon subunits was observed with cupric 1,10-phenanthrolinate treatment. The linkage between the c and gamma subunits was localized to that part of the gamma subunit between residues 202-286, based on peptide analysis. Reference to the high resolution structure of F1 [Abrahams et al. (1994) Nature 370, 621-628] appears to limit this contact site to the region including residues 202-230. This segment contains 4 tyrosines and 1 tryptophan as possible reactive residues for cross-linking with the c subunit cysteine.

Adenosine Triphosphate↗
PubMed1995

Assembly of F0 sector of Escherichia coli H+ ATP synthase. Interdependence of subunit insertion into the membrane.

The F0 sector of the Escherichia coli H+ transporting ATP synthase is composed of a complex of three subunits, each of which traverses the inner membrane. We have studied the interdependence of subunit insertion into the membrane in a series of chromosomal mutants in which the primary mutation prevented insertion of one of the F0 subunits. Subunit insertion was assessed using Western blots of mutant membrane preparations. Subunit b and subunit c were found to insert into the membrane independently of the other two F0 subunits. On the other hand, subunit a was not inserted into membranes that lacked either subunit b or subunit c. The conclusion that subunit a insertion is dependent upon the co-insertion of subunits b and c differs from the conclusion of several studies, where subunits were expressed from multicopy plasmids.

Cell Membrane↗
PubMed1995

Determination of local protein structure by spin label difference 2D NMR: the region neighboring Asp61 of subunit c of the F1F0 ATP synthase.

Purified subunit c from the H(+)-transporting F1F0 ATP synthase of Escherichia coli folds as an antiparallel pair of extended helices in a solution of chloroform-methanol-water. A similar hairpin-like folding is predicted for the native protein in the multisubunit transmembrane Fo sector of the ATP synthase. A single Cys variant (A67C) of subunit c was created and modified with a maleimido-PROXYL [[3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyl]oxy] spin label. Pairs of 1H 2D correlation and NOE spectra were collected with the nitroxide oxidized (paramagnetic) and reduced (diamagnetic). The pairs of spectra were subtracted, yielding difference spectra containing only cross-peaks from 1H within 15 A of the spin label. These greatly simplified spectra were easily analyzed to provide complete assignments for residues 10-25 and 52-79 of the protein, 150 NOE distance restraints, and 27 hydrogen-bonding restraints. The chemical shifts and NOE patterns observed in the derivatized mutant were virtually identical to those which were resolved in the unmodified wild-type protein, strongly suggesting that the spin label was not perturbing the protein structure. The restaints enabled us to calculate a detailed structure for this region of subunit c. The structure consisted of two gently curved helices, crossing at a slight (30 degrees) angle. The C-terminal helix was disrupted from Val60 to Ala62 near the essential Pro64. Asp61, the residue thought to undergo protonation--deprotonation with each H+ transported across the membrane, was in ver der Waals contact with Ala24. The proximity of these residues had been predicted from mutant analyses, where H+ translocation was retained on moving the Asp from position 61 to 24.

Escherichia coli↗
PubMed1995

Changing the ion binding specificity of the Escherichia coli H(+)-transporting ATP synthase by directed mutagenesis of subunit c.

Most F1F0 type ATP synthases, including that in Escherichia coli, use H+ as the coupling ion for ATP synthesis. However, the structurally related F1F0 ATP synthase in Propionigenium modestum uses Na+ instead. The binding site for Na+ residues in the F0 sector of the P. modestum enzyme. We postulated that Na+ might interact with subunit c of F0. Subunit c of P. modestum and E. coli are reasonably homologous (19% identity) but show striking variations around the H(+)-translocating, dicyclohexylcarbodiimide-reactive carboxyl (Asp61 in E. coli). Several hydrophobic residues around Asp61 were replaced with polar residues according to the P. modestum sequence in the hope that the polar replacements might provide liganding groups for Na+. One mutant from 31 different mutation combinations did generate an active enzyme that binds Li+, the combination being V60A, D61E, A62S, and I63T. Li+ binding was detected by Li+ inhibition of ATP-driven H+ transport, Li+ inhibition of F1F0-ATPase activity, and Li+ inhibition of F0-mediated H+ transport. The Li+ effects were observed with membrane vesicles prepared from a delta nhaA, delta nhaB mutant background which lacks Na+/H+ antiporters, and with purified, reconstituted preparations of F0 prepared from this background strain. Li+ inhibition was observed at pH 8.5 but not at pH 7.0. H+ thus appears to compete with Li+ for the binding site. Li+ binding was abolished by replacement of Glu61 by Asp or Ser62 by Ala. The side chains at Ala60 and Thr63 may act in a supporting structural role by providing a more flexible conformation for the Li+ binding cavity. Thr63 does not appear to provide a liganding group since H+ transport in two other mutants, with Gly or Ala in place of Thr63, was also inhibited by Li+. We suggest that a X-Glu-Ser-Y or X-Glu-Thr-Y sequence may provide a general structural motif for monovalent cation binding, and that the flexibility provided by residues X and Y will prove crucial to this structure.

Base Sequence↗
PubMed1994

Suppressor mutations in F1 subunit epsilon recouple ATP-driven H+ translocation in uncoupled Q42E subunit c mutant of Escherichia coli F1F0 ATP synthase.

The Q42E mutation in the polar loop of subunit c of the Escherichia coli F1F0 ATP synthase leads to an uncoupling of H+ translocation through F0 and ATP synthesis/hydrolysis in F1. We have isolated four second-site suppressor mutants in which the coupling defect is corrected. Substitutions for Glu31 in F1 subunit epsilon were found in each suppressor mutant, where the substitutions were E31G, E31V, and E31K (the last being found twice). The different substitutions vary in effectiveness in restoring wild type growth properties in the order epsilon E31G > epsilon E31V > epsilon E31K. Biochemical properties of epsilon E31G/cQ42E and epsilon E31K/cQ42E membranes were compared. In epsilon E31G/cQ42E mutant membranes, ATP-driven H+ translocation by F1F0 and the binding and coupling of F1 to F0 showed a striking pH dependence. Near normal function was observed at pH 7.0, but function was lost at pH 7.8. The function of epsilon E31K/cQ42E membranes was much less affected by changes in pH. Relative to epsilon E31G/cQ42E membranes, the ATP-driven H+ transport function of epsilon E31K/cQ42E membranes was approximately the same at pH 7.5, greater at pH 7.8, and less at pH 7.0. The differences between mutants could be explained if cGlu42 ionized at pH 7.8 with loss of function in epsilon E31G/cQ42E membrane and a similar ionization were compensated for by the positively charged Lys in the epsilon E31K/cQ42E membrane.

Adenosine Triphosphate↗
PubMed1994

Arginine 41 of subunit c of Escherichia coli H(+)-ATP synthase is essential in binding and coupling of F1 to F0.

Two substitutions were made for Arg41 in the polar loop of subunit c of the Escherichia coli F1F0 H(+)-transporting ATP synthase. The R41K and R41H mutants were initially studied by use of a plasmid carrying the complete c R41K or c R41H unc (F1F0) operon in a chromosomal strain deleted for the unc operon. The extent of F0 incorporation into membranes of these cells was quite variable, and the system was concluded to be unsuitable for biochemical characterization. Ultimately, the mutant genes were recombined into the chromosome using a novel method for the unc system. The biochemical phenotype of the chromosomally expressed mutants proved to be reproducible. The c R41H mutation causes a specific defect in assembly of F0, i.e. subunit a was not incorporated into the membrane despite near normal incorporation of subunits b and c. On the other hand, c R41K mutant F0 assembled normally in one of two background strains studied. (In the second genetic background, subunit a was inefficiently incorporated into the c R41K membrane.) In membranes prepared from a c R41K strain assembling a complete F0, R41K F0 was found to bind F1 with near normal affinity and to transport H+ at near normal rates. Although R41K F0 binds F1, F1-ATPase activity and H+ transport remained uncoupled. The uncoupling was indicated by a lack of ATP-driven H+ translocation and by the high proton permeability of membranes with F1 bound to F0. The uncoupled phenotype of the R41K mutant closely resembles that previously reported for the c Q42E mutant.

Adenosine Triphosphate↗
PubMed1994

Essential aspartate in subunit c of F1F0 ATP synthase. Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-1.

Subunit c of the F1F0 type, H(+)-transporting ATP synthase contains an essential Asp that is thought to function in H+ transport. Subunit c folds as a helical hairpin of two transmembrane helices with the essential Asp centered at residue 61 in transmembrane helix-2. Miller et al. (Miller, M. J., Olderburg, M., and Fillingame, R. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4900-4904) have described a functional subunit c variant in which the essential Asp was moved from helix-2 to residue 24 on helix-1 with replacement of Asp61 by Gly. The function of the A24D/D61G subunit c variant is not optimal. In this study, 11 position 61 variants of an A24D subunit c were generated by site-directed mutagenesis in order to test the importance of the position 61 residue. Three functional combinations were found with activities in the order:A24D/D61N > A24D/D61G > or = A24D/D61S. Other substitutions at position 61, including Ala and Cys, did not support function in the A24D protein. Although the A24D/D61N variant showed the highest rates of ATPase-coupled H+ transport, its F0 was inactive in passive H+ transport when F1 was stripped from the membrane. On the other hand, passive H+ transport by A24D/D61G and A24D/D61S stripped membranes approached that of wild type. The defect in function in these two mutants must be ascribed to events related to coupling ATPase and H+ transport. An A24D subunit c (with Asp at both position 24 and 61) was also generated. Its function proved to be pH-dependent. Activity approaching that of wild type was observed at pH 7.0, but function was almost completely lost at pH 7.8. The pH-dependent loss of ATP synthase function led to a slowing of growth on succinate as carbon source on raising the pH from 7.0 to 7.8. In the A24D mutant, with a second Asp at position 61, we postulate that 1 Asp must be protonated before the other can function in H+ transport.

Amino Acid Sequence↗
PubMed1994

Transmembrane helix-helix interactions in F0 suggested by suppressor mutations to Ala24-->Asp/Asp61-->Gly mutant of ATP synthase subunit.

A mutant of ATP synthase subunit c was isolated in which the essential aspartate was exchanged from position 61 on transmembrane helix-2 to position 24 on transmembrane helix-1 (Miller, M. J., Oldenburg, M., and Fillingame, R. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4900-4904). The H+ transporting ATP synthase function of the Ala24-->Asp/Asp61-->Gly mutant is not optimal, and cells grow more slowly than wild type. Twenty-three third-site suppressor mutants with optimized function were isolated in this study. Ten of the optimizing mutations were located to helix-2 of subunit c, and seven of these fell in residues Phe53, Met57, and Met65. The side chains of these three residues are proposed to form a hydrophobic surface on transmembrane helix-2, which participates in the presentation or occlusion of the essential aspartate carboxyl group during proton translocation. The other 13 optimizing mutations were located to subunit a, and 10 of these fell in residues Ala217, Ile221, and Leu224. These three residues are proposed to lie on one face of a transmembrane alpha-helix that includes the essential Arg210 residue. This helix is proposed to interact with the transmembrane bihelical unit of subunit c during protonation and deprotonation of the essential Asp24 in the mutant or Asp61 in wild type.

Alanine↗
PubMed1994

Hairpin folding of subunit c of F1Fo ATP synthase: 1H distance measurements to nitroxide-derivatized aspartyl-61.

Subunit c from the F1Fo ATP synthase of Escherichia coli folds in a hairpinlike structure of two alpha-helices in a solution of chloroform-methanol-H2O, and thus resembles the structure predicted for the folded protein in the membrane. The relevance of the structure in solution to the native structure was demonstrated. Asp61 in the second helical arm was shown to retain its unique reactivity with dicyclohexylcarbodiimide (DCCD) in chloroform-methanol-H2O solution. Further, the protein purified from the Ile28-->Thr DCCD-resistant mutant proved to be less reactive with DCCD in solution. This suggested that the protein folded with Ile28 of the first helical arm close to Asp61 in the second helical arm. Subunit c in wild-type E. coli membranes was specifically labeled with a nitroxide analog of DCCD (NCCD), and the derivative protein was purified. DQF COSY spectra were recorded, and the distances between the paramagnetic nitroxide and resolved protons in the spectra were calculated based upon paramagnetic broadening of the 1H resonances. The paramagnetic contribution to T2 relaxation in the NCCD-labeled sample was calculated by an iterative computer-fitting method, where a control spectrum of a phenylhydrazine-reduced sample was broadened until the line shape of one-dimensional slices through each COSY cross-peak maximally mimicked the line shape of the paramagnetic sample. The distances calculated from paramagnetic broadening indicate that Ala24 and Ala25 in helix-1 lie close (ca. 12 A) to the derivatized Asp61 in helix-2. A model for the interaction of helices in the NCCD-modified protein was generated by restrained molecular mechanics and molecular dynamics using 25 distances of < 10-20 A derived from paramagnetic broadening in combination with 15 long-range nuclear Overhauser enhancement (NOE) restraints (2-5 A) for distances between helices and the 89 intrahelical NOEs that defined helical structure in the DCCD-modified protein.

Aspartic Acid↗