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 α-Subunit

Five monoclonal antibodies against the α subunit of F1-ATPase from Escherichia coli α104, α105, α107, α109, and α110 were prepared. The monoclonal antibodies α104 and α110 enhanced the F1-ATPase activity maximally to 1.6- and 1.7-fold that of the wild-type, respectively, while α105 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 α subunit to the antibodies were identified at residues Ser-440, Leu-456, Leu-471, Leu-482, Met-483, and Ser-506 for α104 and residues Ser-440, Leu-456, Leu-471, Asp-476, Leu-482, Met-483, and Ser-506 for α110. These residues are possibly involved in the epitopes for the antibodies and are located close together on the surface of the α 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 α104 and α110 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.