An additional acidic residue in the membrane portion of the <i>b</i>-subunit of the energy-transducing adenosine triphosphatase of <i>Escherichia coli</i> affects both assembly and function

125

Subunit a of the E. coli F 1 F 0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu 219 and His 245 . A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 238, 243, and 245. The mutants were tested for growth yield, binding of F 1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase activity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F 1 . Significant loss of function was seen only with insertions after positions 238 and 243. In contrast, both insertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type. The other insertions showed an intermediate loss of function. Missense mutations of His 245 to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function. Replacement of Leu 162 by histidine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate. An interaction between Trp 241 and His 245 may be involved in gating a "halfchannel" from the periplasmic surface of F 0 to Asp 61 of subunit a.The F 1 F 0 ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (for recent reviews, see Refs. 1-4). It comprises an F 1 complex, which contains the nucleotide-binding subunits involved in catalysis, and an F 0 complex, which conducts protons across the membrane. The enzyme from E. coli appears to be a minimal form of the ATP synthase, with eight essential subunits. Five different subunits are found in F 1 : ␣, ␤, ␥, ␦, and ⑀, in a stoichiometry of 3:3:1:1:1. Three different subunits, a, b, and c, form F 0 with a likely stoichiometry of 1:2:9 -12 (5). The movement of protons through F 0 , from the periplasm to the cytoplasm, drives the net synthesis of ATP by F 1 .The pathway of protons through F 0 is thought to involve subunits a and c. Subunit b is essential for assembly of a functional F 0 (6, 7) but is not likely to participate directly in proton translocation. The subunits b are embedded in the membrane through a span of about 30 hydrophobic amino acids at the NH 2 terminus. NMR studies of c (8, 9) have confirmed the ␣-helical hairpin structure of the two predicted transmembrane spans and have provided details about the environment of the essential residue Asp 61 (10,11). Questions remain about the oligomeric structure of c subunits and how they interact with subunits a and b. Atomic force microscopy (12, 13) and electron spectroscopic imaging (14) have provided some evidence for a ring of 9 -12 c subunits adjacent to the subunits a and b. Subunit a is the largest of the F 0 subunits (271 residues). Mutagenesis has revealed that in addition to Asp 61 of subunit c, three residues in subunit a seem to be important in proton translocation: Arg 210 , Glu 219 , and His 245 (15)(16)(17)(18)(19). Of these three, only Arg 210 is...

Section: Discussionmentioning

confidence: 99%

Insertion Scanning Mutagenesis of Subunit a of the F1F0 ATP Synthase near His245and Implications on Gating of the Proton Channel

Vik

Patterson

Antonio

1998

125

“…Our present results indicate that the 128 and 128Ј positions are close together in the b syn dimer, and therefore it is likely that the effects of the A128D mutation are due to mutual repulsion of the like-charged residues incorporated at a key interaction site. A similar explanation likely holds true for the observation that a G131D mutation in b prevents proper assembly of the F 1 F 0 complex (14).…”

Section: Discussionmentioning

confidence: 87%

“…Proteolysis studies have shown that the hydrophilic portion of b is required for the binding of F 1 to the membrane (11)(12)(13), and mutation of the glycine at position 131 to aspartate prevents proper assembly of the F 1 F 0 complex (14). Therefore the b subunit provides a critical link between the two sectors, but its role in energy transduction has yet to be determined.…”

mentioning

confidence: 99%

Dimerization Interactions of the b Subunit of the Escherichia coliF1F0-ATPase

McLachlin¹,

Dunn

1997

Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F 1 F 0 -ATPase. Individual cysteine residues were incorporated into b syn , a soluble form of the protein lacking the membrane-spanning Nterminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124. The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the b syn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu 2؉ . Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for b syn . Mixed disulfide formation most readily occurred with the S60C ؉ L65C and A61C ؉ L65C pairs. Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel ␣-helical interaction between the subunits in this region. Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form. These results imply that residues 53-66 but not 24 -52 are essential for b syn dimerization. Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124 -139.The F 1 F 0 -ATP synthase, or ATPase, is responsible in oxidative phosphorylation and photophosphorylation for the translocation of protons across a membrane with concomitant synthesis of ATP. The enzyme complex consists of a membranespanning sector (F 0 ) that conducts protons, and a membraneperipheral sector (F 1 ) that uses energy from proton movement to synthesize ATP (for reviews, see Refs. 1-4). The mechanism by which energy coupling from F 0 to F 1 takes place is currently under intense investigation.In Escherichia coli, the F 0 sector is composed of three different subunits of stoichiometry ab 2 c 9 -12 ; the two b subunits are believed to exist as a dimer. The a and c subunits appear to be directly involved in proton movement across the membrane, while b is not. The arrangement of the F 0 subunits within the membrane is uncertain; some believe that the c subunits are clustered together with a and b on the periphery (5-7) while others propose that a and the two b subunits are surrounded by a ring of c subunits (8, 9). b has a single transmembrane region at its N terminus; the remainder of the 156-residue protein is hydrophilic with the exception of a short stretch of hydrophobic amino acids at residues 124 -132. Based on its sequence the protein is predicted to be largely ␣-helical in nature, and a heptad repeat has been discerned between residues 33 and 79, suggestive of a coiled-coil interaction i...

“…b is thought to exist as a dimer in the complex (5-7), and proteolysis studies have shown that the hydrophilic region of b is required for the association of F 1 with the membrane (7)(8)(9). Removal of two residues from the C terminus of b disrupts normal assembly of the complex (10), as does mutation of Gly-131 to aspartate (11). Thus the b subunit is essential for linking the F 1 and F 0 sectors and likely plays a key role in the coupling of energy from proton translocation to ATP synthesis.…”

mentioning

confidence: 99%

The b and δ Subunits of the Escherichia coli ATP Synthase Interact via Residues in their C-terminal Regions

McLachlin¹,

Bestard

Dunn

1998

An affinity resin for the F 1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b 24 -156 , a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F 1 . Truncated forms of b 24 -156 , in which one or four residues from the C terminus were removed, competed poorly for F 1 binding, suggesting that these residues play an important role in b-F 1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b 24 -156 resulted in a disruption of its association with the purified ␦ subunit of the enzyme. To determine whether these residues interact directly with ␦, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and ␦ were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b 24 -156 -F 1 complex and the membranebound F 1 F 0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue ␦ subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and ␦ subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F 1 sector to the b subunit of F 0 .In the process of oxidative phosphorylation or photophosphorylation, the electron transport chain generates a transmembrane proton gradient. The ATP synthase, or F 1 F 0 -ATPase, allows protons to flow down this electrochemical gradient and uses the energy obtained to synthesize ATP (for reviews, see Refs. 1-4). Under appropriate conditions ATP synthase can hydrolyze ATP to pump protons. The enzyme is composed of two sectors; the F 0 sector is membrane-integral and is responsible for proton translocation, and the F 1 sector is attached to the membrane via F 0 and houses the catalytic sites for ATP synthesis. F 1 is easily detached from the membrane and can be purified as a soluble protein with ATPase activity.ATP synthases contain at least eight types of subunits. In the relatively simple enzyme from Escherichia coli, the F 1 sector has the stoichiometry ␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 , whereas F 0 is composed of three subunits of stoichiometry a 1 b 2 c 9 -12 . The a and c subunits, but not b, contain residues essential for the translocation of protons across the membrane (3). The 156-residue b subunit is believed to span the membrane once at its hydrophobic N terminus, whereas the remainder of the protein is very hydrophilic. b is thought to exist as a dimer in the complex (5-7), and proteolysis studies have shown that the hydrophilic region of b is required for the association of F 1 with the membrane (7-9). Removal of two residues from the C terminus of b disrupts normal assembly of the complex (10), as does mut...