The cytoplasmic loops of subunit a of Escherichia coli ATP synthase may participate in the proton translocating mechanism.
Subunit a plays a key role in promoting H(+) transport and the coupled rotary motion of the subunit c ring in F(1)F(0)-ATP synthase. H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(0) subunit c. H(+) are thought to reach Asp-61 via aqueous pathways mapping to the surfaces of TMHs 2-5 of subunit a based upon the chemical reactivity of Cys substituted into these helices. Here we substituted Cys into loops connecting TMHs 1 and 2 (loop 1-2) and TMHs 3 and 4 (loop 3-4). A large segment of loop 3-4 extending from loop residue 192 loop to residue 203 in TMH4 at the lipid bilayer surface proved to be very sensitive to inhibition by Ag(+). Cys-161 and -165 at the other end of the loop bordering TMH3 were also sensitive to inhibition by Ag(+). Further Cys substitutions in residues 86 and 93 in the middle of the 1-2 loop proved to be Ag(+)-sensitive. We next asked whether the regions of Ag(+)-sensitive residues clustered together near the surface of the membrane by combining Cys substitutions from two domains and testing for cross-linking. Cys-161 and -165 in loop 3-4 were found to cross-link with Cys-202, -203, or -205, which extend into TMH4 from the cytoplasm. Further Cys at residues 86 and 93 in loop 1-2 were found to cross-link with Cys-195 in loop 3-4. We conclude that the Ag(+)-sensitive regions of loops 1-2 and 3-4 may pack in a single domain that packs at the ends of TMHs 3 and 4. We suggest that the Ag(+)-sensitive domain may be involved in gating H(+) release at the cytoplasmic side of the aqueous access channel extending through F(0).
Moore
Kyle J
KJ
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Angevine
Christine M
CM
Vincent
Owen D
OD
Schwem
Brian E
BE
Fillingame
Robert H
RH
D001692:Biological Transport; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D012834:Silver / Q000494:pharmacology; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011522:Protons; D012834:Silver; D006180:Proton-Translocating ATPases; D003545:Cysteine