Monoclonal antibodies recognizing surface residues of the beta subunit of Escherichia coli F(1) ATPase: functional importance of the epitope residues.

Two monoclonal antibodies, beta 208 and beta 210, against the beta subunit of the F(1) ATPase from Escherichia coli reacted with an intact beta subunit and also a peptide corresponding to a portion of beta between residues 1 and 145. Mutations at Ala-1, Val-15, Glu-16, Phe-17, Leu-29, Gly-65, or Leu-66, and His-110 or Arg-111 for beta 210 and beta 208, respectively, caused decreased antibody binding to beta, suggesting that these residues form the epitopes and are thought to lie close together on the surface of the beta subunit. The topological locations of the corresponding residues in the atomic structure of the bovine beta subunit agree well with these expectations, except for Ala-1 and Leu-29. beta 210 binds to two beta strands including the epitope residues that are 50 residues apart, indicating that this antibody recognizes the tertiary structure of the N-terminal end region. Mutations in the epitope residues of beta 210 do not affect the F(1) ATPase activity, suggesting that surfaces of the two beta strands in the amino-terminal end region are not functionally essential. To analyze the functional importance around His-110 recognized by beta 208 we introduced site specific mutations at residues His-110 and Ile-109. Ile-109 to Ala or Arg, and His-110 to Ala or Asp caused defective assembly of F(1). However, the His-110 to Arg mutation had no effect on molecular assembly, suggesting that Ile-109 and His-110, especially the positive charge of His-110 are essential for the assembly of F(1). The His-110 to Arg mutation caused a large decrease in F(1)-ATPase activity, suggesting that a subtle change in the topological arrangement of the positive charge of His-110 located on the surface of beta plays an important role in the catalytic mechanism of the F(1)-ATPase.

Enhancement of Escherichia coli H(+)-ATPase caused by binding of monoclonal antibodies is attributed to structural changes of Leu-456 and Ser-440 in the alpha subunit.

Five monoclonal antibodies against the alpha subunit of F1-ATPase from Escherichia coli alpha 104, alpha 105, alpha 107, alpha 109, and alpha 110 were prepared. The monoclonal antibodies alpha 104 and alpha 110 enhanced the F1-ATPase activity maximally to 1.6- and 1.7-fold that of the wild-type, respectively, while alpha 105 did not. Both antibodies bound to a peptide corresponding to the region between residues 354 and 513. Mutations in this region which caused reduced binding of the alpha subunit to the antibodies were identified at residues Ser-440, Leu-456, Leu-471, Leu-482, Met-483, and Ser-506 for alpha 104 and residues Ser-440, Leu-456, Leu-471, Asp-476, Leu-482, Met-483, and Ser-506 for alpha 110. These residues are possibly involved in the epitopes for the antibodies and are located close together on the surface of the alpha subunit. Among the mutations, Leu-456 to Pro and Ser-440 to Pro mutations caused increase of the F1-ATPase activity up to 1.9 and 1.2 times that of the wild-type, respectively, while Leu-471 to Pro mutation caused a defect in assembly of the F1-ATPase on the membrane. The other mutations caused no significant change in ATPase activity. These results suggested that Ser-440 and Leu-456 have an important role in regulating catalysis by the F1-ATPase, but that the neighboring residue Leu-471 has an important role in assembly of the F1-ATPase complex. It was also suggested that binding of the monoclonal antibodies alpha 104 and alpha 110 to residues Ser-440 and Leu-456 caused local conformational changes, leading to enhancing effects on F1-ATPase activity similar to the Ser-440 to Pro and Leu-456 to Pro mutations.