Trapping of conformations of the Escherichia coli F1 ATPase by disulfide bond formation. A state of the enzyme with all three catalytic sites of equal and low affinity for nucleotides.
A mutant of Escherichia coli F1F0-ATPase, alphaS411C/betaY331W/betaE381C/gammaC87S, has been generated. CuCl2 treatment of this mutant led to cross-linking between alpha and beta subunits in yields of up to 90%. This cross-linking across non-catalytic site interfaces inhibited ATP hydrolysis activity. In the absence of cross-linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 microM, 6 microM and 60 microM, respectively, values that are comparable to wild-type. For MgADP, there was one tight site (0.34 microM) and two sites of lower affinity (each 27 microM), again comparable to wild-type enzyme. After cross-linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (approximately 60 microM). Thus cross-linking fixed all three catalytic sites in the same conformation. Trypsin cleavage experiments showed that cross-linking fixed the epsilon subunit in the ATP+EDTA conformation.
Aggeler
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Institute of Molecular Biology, University of Oregon, Eugene 97403-1229, USA.
Grüber
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Capaldi
R A
RA
D001426:Bacterial Proteins / Q000737:chemistry / Q000648:ultrastructure; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology; D009711:Nucleotides / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000648:ultrastructure*; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry
D001426:Bacterial Proteins; D004220:Disulfides; D009711:Nucleotides; D014357:Trypsin; D006180:Proton-Translocating ATPases