Structural interactions between transmembrane helices 4 and 5 of subunit a and the subunit c ring of Escherichia coli ATP synthase

Abstract

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.

FIGURE 1.

Structure of the four bis-MTS reagents used in this study. The theoretical distances between the sulfurs (S) forming disulfide bonds with Cys side chains is indicated (from Ref. 25).

FIGURE 2.

Bis-MTS reagent catalyzed cross-link formation between Cys at position 248 in subunit a and at positions 62, 63, and 65 in subunit c. Membranes were treated with the indicated bis-MTS reagent according “Experimental Procedures.” The effect of pH on cross-link formation was determined by resuspending membrane pellets in TMG buffer at the indicated pH. Solubilized membranes were analyzed by SDS electrophoresis and Western blots were used to visualize subunit a. The bands corresponding to subunit a and the a–c dimer are indicated. A, aI248C/cA62C; B, aI248C/cI63C; and C, aI248C/cM65C. IA indicates immunoartifact.

FIGURE 3.

Purified a–c dimer formed with M2M bis-MTS cross-linker. Membranes of the mutant aI248C/cM65C were treated with M2M and His-tagged subunit a or a–c dimer was purified as described under “Experimental Procedures.” A single gel was run with duplicate loading of samples to probe with both subunit a antibody and subunit c antibody. β-ME, mercaptoethanol.

FIGURE 4.

Bis-MTS reagent catalyzed cross-link formation between Cys at position 251 in subunit a and positions 59 and 62 in subunit c. Membranes were treated at the pH indicated in A or at pH 8.5 in B. Electrophoresis and Western blotting were carried out as described in the legend to Fig. 2. A, aL251C/cL59C; and B, aL251C/cA62C.

FIGURE 5.

Bis-MTS reagent and Cu²⁺ catalyzed cross-link formation between Cys at position 262 in subunit a and position 55 in subunit c at various pH values. aV262C/cI55C membranes were treated with bis-MTS (A) reagents or 1.5 mm Cu²⁺-phenanthroline (B) as described under “Experimental Procedures.” Electrophoresis and Western blotting were carried out as described in the legend to Fig. 2.

FIGURE 6.

Bis-MTS reagent catalyzed cross-link formation between Cys at position 262 in subunit a and positions 53 and 54 in subunit c. Membranes were treated at pH 8.5 and electrophoresis and Western blotting were carried out as described in the legend to Fig. 2. A, aV262C/cF53C; and B, aV262C/cF54C.

FIGURE 7.

pH-dependent cross-link formation between Cys at position 252 in subunit a and positions 62 and 65 in subunit c with bis-MTS reagents. Membranes were treated and electrophoresis and Western blotting were carried out as described in the legend to Fig. 2. A, aQ252C/cA62C; and B, aQ252C/cM65C.

FIGURE 8.

pH-dependent cross-link formation between Cys at position 263 in subunit a and position 54 in subunit c with bis-MTS reagents. aY263C/cF54C membranes were treated and electrophoresis and Western blotting was carried out as described in the legend to Fig. 2.

FIGURE 9.

A c—a–c trimer is formed by sequential treatment with M3M and Cu²⁺. A, membranes of the aL224C/cF54C and aV262C/cY73C quadruple mutant were treated with Cu²⁺, M3M, or both M3M and Cu²⁺ to catalyze cross-link formation. B, the His-tagged subunit a, a–c, or c—a–c products were purified as described under “Experimental Procedures.” A single gel was run with duplicate loading of samples to probe with both subunit a antibody and subunit c antibody as described in the legend to Fig. 3.

FIGURE 10.

Schematic representation of TMH4 and TMH5 of subunit a docked next to three copies of TMH2 of subunit c based on available cross-linking data. aTMH4 and aTMH5 (yellow cylinders) were placed next to three identically oriented copies of cTMH2, the cTMH2 orientation being based upon the I. tartaricus x-ray structure (7). Residues involved in cross-linking in this study and Jiang and Fillingame (22) are indicated. The schematic illustrates how the helices might pack next to subunit c and indicate one possible means by which aTMH5 and aTMH4 interact with different cTMH2s. The helices are assumed to pack in parallel. aTMH4 and aTMH5 are oriented relative to each other based upon the aG218C/I248C zero-length cross-link catalyzed by Cu²⁺-phenanthroline (21).