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.
Highlights
Cross-linking between aTMH5 and cTMH2—In a previous study from this laboratory, extensive Cu2ϩ-catalyzed crosslinking between Cys substitutions in aTMH4 and cTMH2 was observed over a span of 19 amino acid residues in the two transmembrane helix (TMH) [22]
These results suggest that aTMH4 may pack close to the periphery of the c-ring and that aTMH5 may pack at a greater distance
We reported that aTMH4 could be cross-linked to cTMH2 via Cu2ϩ-catalyzed disulfide bond formation over a span of 19 amino acids, which suggested that these helices packed in close proximity and possibly in parallel to each other [22]
Summary
Interaction between Transmembrane Helices of Subunits a and c position of several sets of second site suppressor mutations, we recently introduced pairs of Cys into putatively apposing TMHs and tested for zero-length cross-linking with disulfide bond formation catalyzed by Cu2ϩ [21]. Cu2ϩ-catalyzed cross-links were observed between Cys pairs introduced into aTMH4 and cTMH2 [22]. Zero-length cross-links between aTMH5 and cTMH2 were not identified in a previous survey of 32 Cys pairs.. In this study we have attempted to determine the distance between aTMH5 and cTMH2 using bis-MTS reagents that react to insert variable length spacers between cross-linkable Cys pairs. Crosslinking was attempted in 62 Cys substituted pairs in aTMH5 and cTMH2 and positive cross-links were observed in 12 cases. Certain cross-links demonstrated a pH dependence that would be consistent with aTMH5 rotating in response to acidification of the local environment We suggest from these results that aTMH5 packs next to the c-ring and could play a role in gating Hϩ transport to cAsp-61 in response to acidification of the periplasmic half-channel in F0
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