Rat liver mitochondrial ATP synthase. Effects of mutations in the glycine-rich region of a β subunit peptide on its interaction with adenine nucleotides

The β subunit of the rat liver mitochondrial ATP synthase contains a glycine-rich amino acid sequence implicated in binding nucleotides by its similarity to a sequence found in many other nucleotide-binding proteins. A C-terminal three-quarter-length rat liver β subunit fragment (Glu¹²² through Ser⁴⁷⁹), containing this homology region, interacts with adenine nucleotides (Garboczi, D.N., Hullihen, J.H., and Pedersen, P.L. (1988) J. Biol. Chem. 263, 15694-15698). Here we directly test the involvement of the glycine-rich region in nucleotide binding by altering its amino acid sequence through mutation or deletion. Twenty-one mutations within the glycine-rich region of the β subunit cDNA were randomly generated. Wild-type and mutant β subunit proteins were purified from overexpressing Escherichia coli strains. The mutant proteins were screened for changes in their interaction with 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), a fluorescent nucleotide analog. Only one mutant protein bearing two amino acid changes (Gly¹⁵³ → Val, Gly¹⁵⁶ → Arg) exhibited a fluorescence enhancement higher than that of the wild-type 'control'. Further analysis of this protein revealed a lower affinity for TNP-ATP (K(d) = 10 μM) compared with wild type (K(d) = 5 μM). In addition, a mutant from which amino acids Gly¹⁴⁹-Lys²¹⁴ had been deleted was prepared. This mutant protein, which lacks the entire glycine-rich region, also displayed a marked reduction in affinity for TNP-ATP (K(d) > 60 μM). Prior addition of 0.5 mM ATP significantly reduced the binding of TNP-ATP to both the double and deletion mutants. The altered interaction of nucleotide with both glycine-rich region mutants points to the involvement of this region in the binding site. Further, this work shows that a β subunit protein that lacks the glycine-rich homology region can still interact with nucleotide, indicating that one or more additional regions of this subunit contribute to the nucleotide binding site.