title: Rat liver mitochondrial ATP synthase. Effects of mutations in the glycine-rich region of a beta subunit peptide on its interaction with adenine nucleotides.
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.
authors: Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
authors: D N
authors: DN
authors: Garboczi
authors: P J
authors: PJ
authors: Thomas
authors: P L
authors: PL
authors: Pedersen
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D005998:Glycine; D007700:Kinetics; D046911:Macromolecular Substances; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D006180:Proton-Translocating ATPases; D005998:Glycine