Transmembrane Helix Of Subunit Research Articles

Structure of the yeast F 1 F o -ATP synthase dimer and its role in shaping the mitochondrial cristae

We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.

Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7 Å resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.

Evidence of the Proximity of ATP Synthase Subunits 6 (a) in the Inner Mitochondrial Membrane and in the Supramolecular Forms of Saccharomyces cerevisiae ATP Synthase

The involvement of subunit 6 (a) in the interface between yeast ATP synthase monomers has been highlighted. Based on the formation of a disulfide bond and using the unique cysteine 23 as target, we show that two subunits 6 are close in the inner mitochondrial membrane and in the solubilized supramolecular forms of the yeast ATP synthase. In a null mutant devoid of supernumerary subunits e and g that are involved in the stabilization of ATP synthase dimers, ATP synthase monomers are close enough in the inner mitochondrial membrane to make a disulfide bridge between their subunits 6, and this proximity is maintained in detergent extract containing this enzyme. The cross-linking of cysteine 23 located in the N-terminal part of the first transmembrane helix of subunit 6 suggests that this membrane-spanning segment is in contact with its counterpart belonging to the ATP synthase monomer that faces it and participates in the monomer-monomer interface.

Acid Residues in the Transmembrane Helices of the Na+-Pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Involved in Sodium Translocation

Vibrio cholerae and many other marine and pathogenic bacteria possess a unique respiratory complex, the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR), which pumps Na(+) across the cell membrane using the energy released by the redox reaction between NADH and ubiquinone. To function as a selective sodium pump, Na(+)-NQR must contain structures that (1) allow the sodium ion to pass through the hydrophobic core of the membrane and (2) provide cation specificity to the translocation system. In other sodium-transporting proteins, the structures that carry out these roles frequently include aspartate and glutamate residues. The negative charge of these residues facilitates binding and translocation of sodium. In this study, we have analyzed mutants of acid residues located in the transmembrane helices of subunits B, D, and E of Na(+)-NQR. The results are consistent with the participation of seven of these residues in the translocation process of sodium. Mutations at NqrB-D397, NqrD-D133, and NqrE-E95 produced a decrease of approximately >or=10-fold in the apparent affinity of the enzyme for sodium (Km(app)(Na+)), which suggests that these residues may form part of a sodium-binding site. Mutation at other residues, including NqrB-E28, NqrB-E144, NqrB-E346, and NqrD-D88, had a strong effect on the quinone reductase activity of the enzyme and its sodium sensitivity, but a weaker effect on the apparent sodium affinity, consistent with a possible role in sodium conductance pathways.

Structural Interactions between Transmembrane Helices 4 and 5 of Subunit a and the Subunit c Ring of Escherichia coli ATP Synthase

Subunit a plays a key role in promoting H+ transport and the coupled rotary motion of the subunit c ring in F1F0-ATP synthase. H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F0 subunit c. H+ are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a. TMH4 of subunit a is thought to pack close to TMH2 of subunit c based upon disulfide cross-link formation between Cys substitutions in both TMHs. Here we substituted Cys into the fifth TMH of subunit a and the second TMH of subunit c and tested for cross-linking using bis-methanethiosulfonate (bis-MTS) reagents. A total of 62 Cys pairs were tested and 12 positive cross-links were identified with variable alkyl length linkers. Cross-linking was achieved near the middle of the bilayer for the Cys pairs a248C/c62C, a248C/ c63C, a248C/c65C, a251C/c57C, a251C/c59C, a251C/c62C, a252C/c62C, and a252C/c65C. Cross-linking was achieved near the cytoplasmic side of the bilayer for Cys pairs a262C/c53C, a262C/c54C, a262C/c55C, and a263C/c54C. We conclude that both aTMH4 and aTMH5 pack proximately to cTMH2 of the c-ring. In other experiments we demonstrate that aTMH4 and aTMH5 can be simultaneously cross-linked to different subunit c monomers in the c-ring. Five mutants showed pH-dependent cross-linking consistent with aTMH5 changing conformation at lower pH values to facilitate cross-linking. We suggest that the pH-dependent conformational change may be related to the proposed role of aTMH5 in gating H+ access from the periplasm to the cAsp-61 residue in cTMH2.

Aqueous Access Pathways in ATP Synthase Subunit a: REACTIVITY OF CYSTEINE SUBSTITUTED INTO TRANSMEMBRANE HELICES 1, 3, AND 5

Subunit a is thought to play a key role in H+ transport-driven rotation of the subunit c ring in Escherichia coli F1F0 ATP synthase. In the membrane-traversing F0 sector of the enzyme, H+ binding and release occurs at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access to surfaces of TMHs 2, 4, and 5 was previously suggested based upon the chemical reactivity of cysteine residues substituted into these helices. Here we have substituted Cys into TMH1 and TMH3 and extended the substitutions in TMH5 to the cytoplasmic surface. One region of TMH3 proved to be moderately Ag+-sensitive and may connect with the Ag+-sensitive region found previously on the periplasmic side of TMH2. A single Cys substitution in TMH1 proved to be both N-ethylmaleimide (NEM)-sensitive and Ag+-sensitive and suggests a possible packing interaction of TMH1 with TMH2 and TMH3. New Ag+- and NEM-sensitive residues were found at the cytoplasmic end of TMH5 and suggest a possible connection of this region to the NEM- and Ag+-sensitive region of TMH4 described previously. From the now complete pattern of TMH residue reactivity, we conclude that aqueous access from the periplasmic side of F0 to cAsp-61 at the center of the membrane is likely to be mediated by residues of TMHs 2, 3, 4, and 5 at the center of a four-helix bundle. Further, aqueous access between cAsp-61 and the cytoplasmic surface is likely to be mediated by residues in TMH4 and TMH5 at the exterior of the four-helix bundle that are in contact with the c-ring.

Glutamates 99 and 107 in Transmembrane Helix III of Subunit I of Cytochrome bd Are Critical for Binding of the Heme b595-d Binuclear Center and Enzyme Activity

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b558 (T187L) or b595-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b595-d center, as reported for heme b595 ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.

Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane.

The role of subunit a in promoting proton translocation and rotary motion in the Escherichia coli F1Fo ATP synthase is poorly understood. In the membrane-bound Fo sector of the enzyme, H+ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access pathways have previously been mapped to surfaces of aTMH4. Here we have substituted Cys into the second and fifth TMHs of subunit a and carried out chemical modification with Ag+ and N-ethylmaleimide to define the aqueous accessibility of residues along these helices. Access to cAsp-61 at the center of the membrane may be mediated in part by Ag+-sensitive residues 248, 249, 251, and 252 in aTMH5. From the periplasmic surface, aqueous access to cAsp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in aTMH2. The Ag+-sensitive residues in TMH2, -4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane. In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2-5. The aqueous access pathways at the interior of subunit a may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to cAsp-61 during the H+ transport cycle.

Primary structure of a novel subunit in ba3-cytochrome oxidase from Thermus thermophilus.

The bax-type cytochrome c oxidase from Thermus thermophilus is known as a two subunit enzyme. Deduced from the crystal structure of this enzyme, we discovered the presence of an additional transmembrane helix "subunit IIa" spanning the membrane. The hydrophobic N-terminally blocked protein was isolated in high yield using high-performance liquid chromatography. Its complete amino acid sequence was determined by a combination of automated Edman degradation of both the deformylated and the cyanogen bromide cleaved protein and automated C-terminal sequencing of the native protein. The molecular mass of 3,794 Da as determined by MALDI-MS and by ESI requires the N-terminal methionine to be formylated and is in good agreement with the value calculated from the formylmethionine containing sequence (3,766.5 Da + 28 Da = 3,794.5 Da). This subunit consits of 34 residues forming one helix across the membrane (Lys5-Ala34), which corresponds in space to the first transmembrane helix of subunit II of the cytochrome c oxidases from Paracoccus denitrificans and bovine heart, however, with opposite polarity. It is 35% identical to subunit IV of the ba3-cytochrome oxidase from Natronobacterium pharaonis. The open reading frame encoding this new subunit IIa (cbaD) is located upstream of cbaB in the same operon as the genes for subunit I (cbaA) and subunit II (cbaB).

Genetic Fusions of Subunit c in the F0Sector of H+-transporting ATP Synthase: FUNCTIONAL DIMERS AND TRIMERS AND DETERMINATION OF STOICHIOMETRY BY CROSS-LINKING ANALYSIS

The multicopy c subunit of the H+-transporting ATP synthase of Escherichia coli folds through the transmembrane F0 sector as a hairpin of two hydrophobic alpha-helices with the proton-translocating aspartyl-61 side chain centered in the second transmembrane helix. The number of subunits c in the F0 complex, which is thought to determine the H+-pumping/ATP stoichiometry, was previously not determined with exactness but thought to range from 9-12. The studies described here indicate that the exact number is 12. Based upon the precedent of the subunit c in vacuolar-type ATPases, which are composed of four transmembrane helices and seem to have evolved by gene duplication of an F0-type progenitor gene, we constructed genetically fused dimers and trimers of E. coli subunit c. Both the dimeric and trimeric forms proved to be functional. These results indicate that the total number of subunit c in F0 should be a multiple of 2 and 3. Based upon a previous study in which the oligomeric organization of c subunits in F0 was determined by cross-linking of Cys-substituted subunits (Jones, P. C. , Jiang, W., and Fillingame, R. H. (1998) J. Biol. Chem. 273, 17178-17185), we introduced Cys into the first and last transmembrane helices of subunit c monomers, dimers, and trimers and attempted to generate cross-linked products by oxidation with Cu(II)-(1,10-phenanthroline)2. Double Cys substitutions at two sets of positions gave rise to extensive cross-linked multimers. Multimers of the monomer that extended up to the position of c12 were correlated and calibrated with distinct cross-linked species of the appropriate doubly Cys-substituted dimers (i.e. c2, c4, . c12) and doubly Cys-substituted trimers (i.e. c3, c6, c9, c12). The results show that there are 12 copies of subunit c per F0 in E. coli, the exact number having both mechanistic and structural significance.

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.

Cytochrome c oxidase in Neurospora crassa contains myristic acid covalently linked to subunit 1.

Radiolabel from [3H]myristic acid was incorporated by Neurospora crassa into the core catalytic subunit 1 of cytochrome c oxidase (EC 1.9.3.1), as indicated by immunoprecipitation. This modification of the subunit, which was specific for myristic acid, represents an uncommon type of myristoylation through an amide linkage at an internal lysine, rather than an N-terminal glycine. The [3H]myristate, which was chemically recovered from the radiolabeled subunit peptide, modified an invariant Lys-324, based upon analyses of proteolysis products. This myristoylated lysine is found within one of the predicted transmembrane helices of subunit 1 and could contribute to the environment of the active site of the enzyme. The myristate was identified by mass spectrometry as a component of mature subunit 1 of a catalytically active, purified enzyme. To our knowledge, fatty acylation of a mitochondrially synthesized inner-membrane protein has not been reported previously.

Helical structure and folding of subunit c of F1F0 ATP synthase: 1H NMR resonance assignments and NOE analysis.

Subunit c of the H(+)-transporting F1F0 ATP synthase (EC 3.6.1.34) is thought to fold across the membrane as a hairpin of two alpha-helices and function as a key component of the H(+)-translocase of F0. We report here the initial results of a structural study of purified subunit c in a chloroform-methanol-water (4:4:1) solvent mixture using standard two-dimensional NMR techniques. The spin systems of 78 of the 79 amino acid side chains have been assigned to residue type, and 44 of these have been assigned to specific residues in the sequence. Stretches of alpha-helical secondary structure were observed for Asp7-ILe26 in the first proposed transmembrane helix, and for Arg50-Ile55 and Ala67-Val78 in the second proposed transmembrane helix. Nuclear Overhauser effects (NOEs) were observed between residues at both ends of the predicted transmembrane helices. The intensities of the NOEs between helix-1 and helix-2 were not diminished by mixing of 2H-subunit c with 1H-subunit c, and therefore the NOEs must be due to intramolecular, rather than intermolecular, interactions. Hence the purified protein must fold as a hairpin in this solvent system, just as it is thought to fold in the lipid bilayer of the membrane. In native F0, dicyclohexylcarbodiimide reacts specifically with Asp61 in the second transmembrane helix of subunit c, and the rate of this reaction is reduced by substitution of Ile28 by Thr on the first transmembrane helix. The I28T substitution is shown here to alter the chemical shifts of protons at and around Asp61. This observation provides a further indication that subunit c may fold in chloroform-methanol-water solvent much like it does in the membrane.

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.

Mutations in three of the putative transmembrane helices of subunit a of the Escherichia coli F 1F 0-ATPase disrupt ATP-driven proton translocation

Three missense mutants in subunit a of the Escherichia coli F 1F 0-ATPase were isolated and characterized after hydroxylamine mutagenesis of a plasmid carrying the uncB (subunit a) gene. The mutations resulted in Asp 119 → His, Ser 152 → Phe, or Gly 197 → Arg substitutions in subunit a Function was not completely abolished by any of the mutations. The F 0 membrane sector was assembled in all three cases as judged by restoration of dicyclohexylcarbodiimide sensitivity to the F 1F 0-ATPase. The H + translocation capacity of F 0 was reduced in all three mutants. ATP-driven H +-translocation was also reduced, with the response in the Gly 197 → Arg mutant being almost nil and that in the Asp 119 → His and Ser 152 → Phe mutants less severely affected. The substituted residues are predicted to lie in the second, third, and fourth transmembrane helices suggested in most models for subunit a. The Gly 197 → Arg mutation lies in a very conserved region of the protein and the substitution may disrupt a structure that is critical to function. The Asp 119 → His and Ser 152 → Phe mutations also lie in areas with sequence conservation. A further analysis of randomly generated mutants may provide more information on regions of the protein that are crucial to function. Heterodiploid transformants, carrying plasmids with either the wild-type uncB gene or mutant uncB genes in an uncB (Trp 231 → stop) background, were characterized biochemically. The truncated subunit a was not detected in membranes of the background strain by Western blotting, and the uncB + plasmid complemented strain showed normal biochemistry. The uncB mutant genes were shown to cause equivalent defects in either the heterodiploid background configuration, or after incorporation into an otherwise wild-type unc operon. The subunit a (Trp 231 → stop) background strain was shown to bind F 1-ATPase nearly normally despite lacking subunit a in its membrane.

Where is the oxygen binding site of cytochrome c oxidase? Transmembrane helices of subunits I and II

Where is the oxygen binding site of cytochrome c oxidase? Transmembrane helices of subunits I and II