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

Abstract

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...

Site-directed Cross-linking of b to the α, β, anda Subunits of the Escherichia coli ATP Synthase

“…Because b has a single membrane-spanning region at its N terminus, the remainder of the subunit must span a distance of over 100 Å to come in contact with ␦. Consistent with this proposed arrangement, the region of interaction between b and ␦ has been localized to the C terminus of b (10,16). The hydrophilic domain of b by itself is mostly ␣-helical as measured by circular dichroism (17), and an isolated complex composed of ␦ with the hydrophilic domain of b was demonstrated by sedimentation velocity ultracentrifugation to be highly extended (9).…”

“…Cross-linking of bA92C to F 1 in membrane preparations was also carried out essentially as described (10), with the following exceptions. Membranes from which F 1 had been removed were diluted to 1 mg of total protein/ml, and then BPM (Molecular Probes, Eugene, OR) was added as described.…”

“…Chemical Cross-linking of Membranes-Cross-linking of bI109C and bE110C to F 1 in membrane preparations was carried out as described previously (10), except that 0.5 mM p-azidophenacyl bromide (APB) (Sigma) dissolved in methanol was used instead of 1 mM benzophenone-4-maleimide (BPM). Cross-linking of bA92C to F 1 in membrane preparations was also carried out essentially as described (10), with the following exceptions.…”

“…In recent years the interaction of the b dimer and ␦ has been well established by a variety of evidence (7)(8)(9)(10). The ␦ subunit appears to be located near the crown of the F 1 complex, the part of F 1 furthest from the membrane (11)(12)(13)(14)(15).…”

“…Due to the relatively weak interaction between the b and ␦ subunits (K D ϭ 5-10 M; Ref. 9), and the apparent structural flexibility of the hydrophilic domain of b (6,10,19), it seems necessary that the binding of b to F 1 must be stabilized through additional interactions. Rodgers and Capaldi (20) have reported the formation of a disulfide bond between a cysteine residue introduced at the C-terminal position of the 156-residue b subunit and the endogenous Cys-90 of ␣.…”