Abstract
We have previously shown that the E31C-substituted epsilon subunit of F1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F0 in the Escherichia coli F1F0-ATP synthase complex (Zhang, Y., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 24609-24614). The interactions of subunits epsilon and c are thought to be central to the coupling of H+ transport through F0 to ATP synthesis in F1. To further define the domains of interaction, we have introduced additional Cys into subunit epsilon and subunit c and tested for cross-link formation following sulfhydryl oxidation. The results show that Cys, in a continuous stretch of residues 26-33 in subunit epsilon, can be cross-linked to Cys at positions 40, 42, and 44 in the polar loop region of subunit c. The results are interpreted, and the subunit interaction is modeled using the NMR and x-ray diffraction structures of the monomeric subunits together with information on the packing arrangement of subunit c in a ring of 12 subunits. In the model, residues 26-33 form a turn of antiparallel beta-sheet which packs between the polar loop regions of adjacent subunit c at the cytoplasmic surface of the c12 oligomer.
Highlights
The relation of structure and mechanism in F0 is less thoroughly understood
The surface of subunit ⑀ lying proximal to subunit c has been mapped by cross-linking experiments to a region encompassing residues 26 –33, which in the NMR and x-ray diffraction structures of subunit ⑀ [38, 39] reside in a loop of antiparallel -sheet (Fig. 5A)
Residue 31 of ⑀ must lie proximal to the surface of F0 because the ⑀E31C-substituted protein can be cross-linked to Cys at positions 40, 42, and 43 of subunit c [25]
Summary
Mutant Construction and Expression—The plasmids constructed in this study are derivatives of plasmid pYZ201 [25], which carries the eight structural genes of unc operon coding F1F0 (bases 870 –10172; Ref. 40). The Cys substitutions in subunit c and subunit ⑀ were introduced by oligonucleotide-directed mutagenesis using the strategy described previously [25]. Mutant Construction and Expression—The plasmids constructed in this study are derivatives of plasmid pYZ201 [25], which carries the eight structural genes of unc operon coding F1F0 (bases 870 –10172; Ref. 40).. Antibodies that nonspecifically cross-reacted with E. coli membrane proteins were removed by preabsorption with membranes prepared from a mutant strain with a deleted unc operon [44]. Structural Modeling of Subunit-Subunit Interaction—A model for subunit c interaction in the c12 oligomer has been derived from the NMR model [27] using distance constraints derived from the cross-linking data of Jones et al [28].3. A somewhat wider distance constraint range of 4 –11 Å was used in the molecular mechanics calculations done here to allow for possible thermal motions and distortions of structure on cross-link formation. Energy minimization was performed with CVFF (constant valence force field) using the steepest descents and conjugated gradient methods as implemented in DISCOVER 3.0 (Molecular Simulations Inc.) until the maximum derivative was below 0.1 kcal molϪ1 ÅϪ1.4
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