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

Abstract

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.

Figure 1

Growth (function) of Cys-substituted mutants (A) and summary of a–c cross-linking results (B). (A) Cells were plated on succinate minimal medium and the colony size (mm) was measured after incubation at 37°C for 3 days as a measure of oxidative phosphorylation function. The chromosomal, wild-type control strain showed colonies of 2 mm after 3 days at 37°C. (B) Relative yield of a–c cross-linked dimer formed between Cys at the positions indicated. Membrane vesicles were treated with CuP at 10°C for 60 min. Relative yield of the a–c cross-link product: 0, none; −/+, ≤5%; +, 6–10%; ++, 11–20%; +++, 20–40%; ++++, >40%. 0* indicates Cys pair forming no cross-link at 10°C but significant cross-link (8%) at 20°C.

Figure 2

Cross-linking of aI221C-substituted membranes in varying combinations with second Cys in subunit c. SDS-solubilized membranes were electrophoresed under nonreducing condition before immunoblotting. The blot was first probed with antiserum against subunit c. The blot was then stripped of bound antibodies by submerging it in a buffer containing 62.5 mM Tris⋅HCl (pH 6.8), 2% SDS, and 100 mM 2-mercaptoethanol for 30 min at 50°C and reprobed with antiserum against subunit a. The bands marked IA* are immunoblotting artifacts. Wild-type membranes are from pDF163- (uncBEFH⁺) transformed strain JWP109 (ΔuncB–H). Deletion membranes are from strain JWP109 (ΔuncB–H).

Figure 3

Comparison of a–c cross-link formation at 10 and 20°C with a series of doubly Cys-substituted membranes. A portion of an immunoblot of a SDS gel run under nonreducing conditions is shown following probing with antiserum to subunit a. The positions of the double Cys substitutions and temperature (10°C or 20°C) of the 1-h CuP cross-linking reaction are indicated.

Figure 4

Formation of a–c cross-links by CuP reaction at 10°C in a series of doubly Cys-substituted membranes. The conditions are as described in Fig. 3. The positions of the double Cys substitutions are indicated. Wild-type membranes are from a pDF163 transformant of strain JWP109 (ΔuncB–H). Deletion membranes are from strain JWP109 (ΔuncB–H).

Figure 5

Summary of major cross-links formed between helix-4 of subunit a and helix-2 of subunit c at 10°C. The circles numbered without specifying residue type indicate the position of the Cys substitutions in α-helical representations of the transmembrane segments. The positions of essential residues aR210 and cD61 are also indicated.

Figure 6

Ribbon depiction of the folding of subunit c derived from the NMR structure. The “front” and “back” face of two subunits c are indicated. A functional c₂ dimer is proposed to be formed by packing the front face of one monomer against the back face of a second monomer (46), i.e., with D61 of one monomer packed between A24 and A62 of a second monomer. The positions of the backbone atoms of key residues discussed in the text are indicated. The illustration was done in the program molmol (45).