Structural interactions between transmembrane helices 4 and 5 of subunit a and the subunit c ring of Escherichia coli ATP synthase.
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.
Moore
Kyle J
KJ
Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706, USA.
Fillingame
Robert H
RH
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
D021122:Protein Subunits; D000251:Adenosine Triphosphatases; D003545:Cysteine
J Biol Chem. 2006 Dec 8;281(49):37861-7
17035244
J Biol Chem. 1998 Jul 3;273(27):17178-85
9642286
J Biol Chem. 1995 Feb 10;270(6):2815-7
7852354
J Biol Chem. 2007 Mar 23;282(12):9001-7
17234633
J Biol Chem. 1997 Dec 19;272(51):32635-41
9405480
J Biol Chem. 1985 Apr 25;260(8):4807-14
2859283
J Biol Chem. 1998 Jun 26;273(26):16235-40
9632682
J Biol Chem. 1995 Dec 8;270(49):29407-12
7493977
Physiol Rev. 1988 Jan;68(1):177-231
2892214
Science. 2005 Apr 29;308(5722):659-62
15860619
J Biol Chem. 2008 May 2;283(18):12365-72
18332132
Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350-4
388439
Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6607-12
9618459
Nat Rev Mol Cell Biol. 2001 Sep;2(9):669-77
11533724
Biochemistry. 1986 Sep 23;25(19):5595-601
3778876
Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4966-71
11320246
J Biol Chem. 1999 Jun 11;274(24):17353-7
10358096
Methods Mol Biol. 1996;57:203-15
8850007
J Biol Chem. 1998 Jun 26;273(26):16241-7
9632683
Proc Natl Acad Sci U S A. 2004 Aug 17;101(33):12159-64
15302927
J Bioenerg Biomembr. 2000 Aug;32(4):365-71
11768298
Trends Biochem Sci. 2002 Mar;27(3):154-60
11893513
Proc Natl Acad Sci U S A. 1999 Jul 6;96(14):7785-90
10393899
Biochim Biophys Acta. 2008 Jan;1777(1):32-8
18068111
FEBS Lett. 2003 Nov 27;555(1):29-34
14630314
Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13179-83
14595019
EMBO Rep. 2006 Mar;7(3):276-82
16607397
EMBO Rep. 2005 Nov;6(11):1040-4
16170308
J Biol Chem. 2001 Oct 5;276(40):36877-80
11518701
J Biol Chem. 2003 Feb 21;278(8):6066-74
12473663