Amino acid replacements at binding sites of monoclonal antibody in the F1-ATPase beta subunit from Escherichia coli caused altered subunit interactions.

The monoclonal antibodies (mAb), beta 12 and beta 31, against the beta subunit of Escherichia coli F1-ATPase (Miki, J., Matsuda, T., Kariya, H., Ohmori, H., Tsuchiya, T., Futai, M., and Kanazawa, H. (1992) Arch. Biochem. Biophys. 294, 373-381) recognize the amino-terminal portion of the maximal 104 residues that are not exposed to the surface of the F1-ATPase. To identify the epitope residues within these 104 residues, we introduced random mutations in the region, and clones without binding activity to the mAb, beta 12 and beta 31, were screened. beta subunits defective in binding with mAb beta 12 or beta 31 had amino acid replacements at residues 26, 40, 52, 71, and 74 or at residues 40, 41, 43, 44, 46, and 52, respectively. These residues could be part of the epitope and are possibly located close together. We tested the effects of the mutations on oxidative phosphorylation-dependent growth by introducing expression plasmids of the beta subunit gene with the point mutations into the beta-less mutant, JP17. The alpha and beta subunits were missing from the JP17 membranes, and both subunits were assembled functionally after expression of the beta subunit was induced by introducing the expression plasmid. The replacement of Leu-40 by Pro caused the amounts of the alpha and beta subunits on the membranes to be reduced less than 20% of the amounts in wild type. The replacement of Glu-41 by Lys caused a loss of the alpha subunit on the membranes, without any decrease in the beta subunit. These results indicated that the mutation of Leu-40 to Pro affects the essential role of the beta subunit in the assembly of the alpha and beta subunits on the membranes and that the mutation of Glu-41 to Lys partly affects it. The amino-terminal region of the beta subunit, in particular its ternary structure, including residues 40 and 41, plays an important role in the molecular assembly of the alpha and beta subunits on the membranes.