Abstract
The beta 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 beta 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 beta subunit cDNA were randomly generated. Wild-type and mutant beta 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 (Kd = 10 microM) compared with wild type (Kd = 5 microM). 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 (Kd greater than 60 microM). 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 beta 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.
Highlights
EFFECTS OF MUTATIONS IN THE GLYCINE-RICH REGION OF A p SUBUNIT PEPTIDE ON ITS INTERACTION WITH ADENINE NUCLEOTIDES*
We directly test the involvement of the glycine-rich region in nucleotide binding by altering its amino acid sequence through mutation or deletion
Synthase @subunit support the hypothesis that the glycinerich region of the p subunit, as well, is involved in or is close to the nucleotide binding site. In this laboratory, we have shown that a 50-amino acid synthetic peptide from the,8 subunit sequence that includes the glycine-rich region interacts with both ATP and an ATP
Summary
EFFECTS OF MUTATIONS IN THE GLYCINE-RICH REGION OF A p SUBUNIT PEPTIDE ON ITS INTERACTION WITH ADENINE NUCLEOTIDES*. Chemical and genetic modification studies of the ATP synthase @subunit support the hypothesis that the glycinerich region of the p subunit, as well, is involved in or is close to the nucleotide binding site (reviewed in Ref. 1) In this laboratory, we have shown that a 50-amino acid synthetic peptide from the ,8 subunit sequence that includes the glycine-rich region interacts with both ATP and an ATP analog, TNP-ATP’ [16]. ) derived from the construction of the C4 expression plasmid (Ref. 17); PMSF, phenylmethylsulfonyl fluoride; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid; mutant “105,” C4 protein hearing two amino acid changes (GIY’~~ + Val, G~Y’~~ + Arg) in the glycine-rich region; SDS, sodium dodecyl sulfate; FDNP-ATP, 3’-O-
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