Rat liver mitochondrial ATP synthase

Effects of mutations in the glycine-rich region of a β subunit peptide on its interaction with adenine nucleotides

David N. Garboczi, Philip J. Thomas, Peter L. Pedersen

Research output: Contribution to journalArticle

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Abstract

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 (Kd = 10 μM) compared with wild type (Kd = 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 (Kd > 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.

Original languageEnglish (US)
Pages (from-to)14632-14637
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number24
StatePublished - Aug 25 1990

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Mitochondrial Proton-Translocating ATPases
Adenine Nucleotides
Liver
Glycine
Rats
Nucleotides
Peptides
Mutation
Mutant Proteins
Adenosine Triphosphate
Adenosine
Amino Acids
Protein Subunits
Amino Acid Sequence
Bearings (structural)
Binding Sites
Sequence Deletion
Escherichia coli
Carrier Proteins
Complementary DNA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rat liver mitochondrial ATP synthase : Effects of mutations in the glycine-rich region of a β subunit peptide on its interaction with adenine nucleotides. / Garboczi, David N.; Thomas, Philip J.; Pedersen, Peter L.

In: Journal of Biological Chemistry, Vol. 265, No. 24, 25.08.1990, p. 14632-14637.

Research output: Contribution to journalArticle

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