title: 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.
authors.affiliation: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Moore
authors.forename: Robert H
authors.initials: RH
authors.lastname: Fillingame
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D000251:Adenosine Triphosphatases; D003545:Cysteine
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