Identification of the F1-binding Surface on the δ-Subunit of ATP Synthase

Abstract

The stator function in ATP synthase was studied by a combined mutagenesis and fluorescence approach. Specifically, binding of ␦-subunit to ␦-depleted F 1 was studied. A plausible binding surface on ␦-subunit was identified from conservation of amino acid sequence and the high resolution NMR structure. Specific mutations aimed at modulating binding were introduced onto this surface. Affinity of binding of wild-type and mutant ␦-subunits to ␦-depleted F 1 was determined quantitatively using the fluorescence signals of natural ␦-Trp-28, inserted ␦-Trp-11, or inserted ␦-Trp-79. The results demonstrate that helices 1 and 5 in the N-terminal domain of the ␦-subunit provide the F 1 -binding surface of ␦. Unexpectedly, mutations that impaired binding between F 1 and ␦ were found to not necessarily impair ATP synthase activity. Further investigation revealed that inclusion of the soluble cytoplasmic domain of the b subunit substantially enhanced affinity of binding of ␦-subunit to F 1 . The new data show that the stator is "overengineered" to resist rotor torque during catalysis.ATP synthase is the membrane enzyme responsible for ATP synthesis in oxidative and photophosphorylation of prokaryotes and eukaryotes, and also for ATP-driven proton pumping to generate the transmembrane proton gradient in bacterial membranes. In Escherichia coli, ATP synthase consists of a complex of eight subunits, ␣ 3 ␤ 3 ␥␦⑀ab 2 c n . It was defined in earlier times in terms of a membrane-peripheral F 1 sector (␣ 3 ␤ 3 ␥␦⑀) containing three catalytic sites, and a membrane-embedded F 0 sector (ab 2 c n ), which carries out transmembrane proton transport. Recent work has demonstrated that the enzyme functions as a rotary motor. The centrally located "rotor" consists of subunits ␥⑀c n . At the top, it rotates inside the ␣ 3 ␤ 3 hexagon, and thus modifies the activities of the catalytic sites; at the base, it rotates against subunit a, thereby facilitating proton movement. In this way, the energy of the proton gradient is transduced into the energy of ATP synthesis/hydrolysis. Understanding the mechanism by which this occurs is currently of major interest. To ensure that subunits a and ␣ 3 ␤ 3 remain firmly fixed in relation to each other they are connected by a peripheral structure, the "stator" stalk, consisting of subunits b 2 and ␦. For recent reviews of the structure and function of ATP synthase, see Refs. 1-3.The stator must be able to resist strain resulting from rotor torque, thus its construction is of considerable importance. The dimer of b-subunits forms an elongated helical connection between subunit a and the C-terminal domain of the ␦-subunit, it lies at one side of the ␣ 3 ␤ 3 hexagon, and its functional domains have been well characterized (4 -6). There may exist functional interactions between b 2 and the ␣ 3 ␤ 3 hexagon (7). Currently only partial high-resolution structure has been reported for the b subunit (8, 9). The ␦-subunit has been shown by electron microscopy to bind to the very top ("crown") of the ␣ 3 ␤ 3 hexagon (10),...

Escherichia coli F1Fo-ATP Synthase with a b/δ Fusion Protein Allows Analysis of the Function of the Individual b Subunits

“…The b type subunit, which interacts primarily with ␦, is close to a so that it can be cross-linked (34); however, the additional binding energy due to this interaction is not required. Such an arrangement would be in line with previous results that suggest that the wild-type stator stalk is "overengineered" to ensure efficient coupling of proton translocation and catalysis (35). Fig.…”

Assembly of the Stator in Escherichia coli ATP Synthase. Complexation of α Subunit with Other F1 Subunits Is Prerequisite for δ Subunit Binding to the N-Terminal Region of α

“…Initially (7) it was shown by proteolysis experiments using the Escherichia coli enzyme that δ subunit bound to the N-terminal region (residues 1-19) of the α subunit in the F 1 -complex. More recently, quantitative binding experiments showed that this interaction is extremely tight (8,9), and utilizes a binding surface formed between two parallel helices on the N-terminal domain of the δ subunit (10). A 22-residue peptide which mimicked residues 1-22 of α subunit was found to bind with high affinity to the same surface on δ subunit (11), forming a 1/1 complex.…”

The rotary mechanism of the ATP synthase

“…In E. coli, subunit a interacts with the b subunits mostly through their single transmembrane segments and parts near the membrane [14,15], while the δ subunit (the ortholog of the mitochondrial OSCP or oligomycin sensitivity conferral protein) interacts with the amino termini of the α subunits at the "top" of the complex, which is the side away from the membrane ( Fig. 1; [16,17]; reviewed in [10,13]). The affinities between the F 1 and δ subunit, and a and b subunits appear to be sufficiently strong to withstand the considerable torque that has been measured in the single particle experiments [6].…”