Mutational analysis of the consensus nucleotide binding sequences in the rat liver mitochondrial ATP synthase β-subunit

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

Research output: Contribution to journalArticle

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Abstract

The coupling step in the biosynthesis of ATP in biological systems is generally believed to involve an energy-requiring release of ATP bound to the β-subunit of the ATP synthase complex. A molecular description of the ATP binding site on the β-subunit is, therefore, critical to understanding the mechanism of coupling in the enzyme. Previously, we reported that a purified, bacterially expressed rat liver β-subunit binds adenine nucleotides tightly and specifically (Garboczi, D. N., Hullihen, J. H., and Pedersen, P. L. (1988) J. Biol. Chem. 263, 15694-15698). In order to assess the contribution of various regions of the isolated β-subunit to the ATP binding site we have systematically deleted four different regions: the N-terminal region, the Walker A consensus region, the Walker B consensus region (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. (1982) EMBO J. 1, 945-951), and a "C" region, which, like the A and B regions, bears homology to adenylate kinase. Plasmids directing the expression of double deletions of A and B regions, and B and C regions were also constructed. In addition, 2 residues outside of these regions, His-177 and Tyr-345, which have been predicted to play a central role in nucleotide binding, were mutated. Rabbit antisera to synthetic peptides of the A and C regions verified the identity of the bacterially expressed mutant proteins. Seven of the eight mutant proteins overexpressed in Escherichia coli were resistant to E. coli proteases in the preparative stages, as predicted for compact folded proteins. Furthermore, circular dichroism spectropolarimetry revealed no profound structural alterations in the purified mutant proteins. Relative to the over-expressed full-length β-subunit, the mutant lacking the A consensus region suffered a 30-fold loss of affinity for ATP and a loss of specificity for 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) over 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate. The mutant proteins lacking either the N-terminal region or the B region exhibited nucleotide binding properties similar to the full-length β-subunit, whereas the mutant protein lacking the C region suffered an order of magnitude reduction in affinity for ATP. The affinity of the A and B region double deletion was indistinguishable from the A region deletion in regard to TNP-ATP binding, while the double deletion mutant lacking the B and C regions was not stably expressed in the E. coli SE6004. Mutant proteins bearing the single-site changes, His-177 → Gln or Tyr-345 → Cys, exhibited nearly identical binding properties to that of the full-length β-subunit. This is the first study in which regions of the isolated β-subunit of an ATP synthase complex have been systematically assessed by mutational analysis for their contribution to ATP binding. The results indicate that the A consensus sequence contributes over one-half of the total binding energy, with the C region making a significant contribution. The N-terminal region, the B region, Tyr-345, and His-177 appear to play little or no role in ATP binding.

Original languageEnglish (US)
Pages (from-to)20331-20338
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number28
StatePublished - Oct 5 1992

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Mitochondrial Proton-Translocating ATPases
Liver
Rats
Nucleotides
Adenosine Triphosphate
Mutant Proteins
Adenosine
Escherichia coli
Bearings (structural)
Binding Sites
Adenylate Kinase
Adenine Nucleotides
Biosynthesis
Dichroism
Biological systems
Binding energy
Consensus Sequence
Adenosine Monophosphate
Immune Sera
Circular Dichroism

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutational analysis of the consensus nucleotide binding sequences in the rat liver mitochondrial ATP synthase β-subunit. / Thomas, Philip J.; Garboczi, David N.; Pedersen, Peter L.

In: Journal of Biological Chemistry, Vol. 267, No. 28, 05.10.1992, p. 20331-20338.

Research output: Contribution to journalArticle

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T1 - Mutational analysis of the consensus nucleotide binding sequences in the rat liver mitochondrial ATP synthase β-subunit

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N2 - The coupling step in the biosynthesis of ATP in biological systems is generally believed to involve an energy-requiring release of ATP bound to the β-subunit of the ATP synthase complex. A molecular description of the ATP binding site on the β-subunit is, therefore, critical to understanding the mechanism of coupling in the enzyme. Previously, we reported that a purified, bacterially expressed rat liver β-subunit binds adenine nucleotides tightly and specifically (Garboczi, D. N., Hullihen, J. H., and Pedersen, P. L. (1988) J. Biol. Chem. 263, 15694-15698). In order to assess the contribution of various regions of the isolated β-subunit to the ATP binding site we have systematically deleted four different regions: the N-terminal region, the Walker A consensus region, the Walker B consensus region (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. (1982) EMBO J. 1, 945-951), and a "C" region, which, like the A and B regions, bears homology to adenylate kinase. Plasmids directing the expression of double deletions of A and B regions, and B and C regions were also constructed. In addition, 2 residues outside of these regions, His-177 and Tyr-345, which have been predicted to play a central role in nucleotide binding, were mutated. Rabbit antisera to synthetic peptides of the A and C regions verified the identity of the bacterially expressed mutant proteins. Seven of the eight mutant proteins overexpressed in Escherichia coli were resistant to E. coli proteases in the preparative stages, as predicted for compact folded proteins. Furthermore, circular dichroism spectropolarimetry revealed no profound structural alterations in the purified mutant proteins. Relative to the over-expressed full-length β-subunit, the mutant lacking the A consensus region suffered a 30-fold loss of affinity for ATP and a loss of specificity for 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP) over 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-monophosphate. The mutant proteins lacking either the N-terminal region or the B region exhibited nucleotide binding properties similar to the full-length β-subunit, whereas the mutant protein lacking the C region suffered an order of magnitude reduction in affinity for ATP. The affinity of the A and B region double deletion was indistinguishable from the A region deletion in regard to TNP-ATP binding, while the double deletion mutant lacking the B and C regions was not stably expressed in the E. coli SE6004. Mutant proteins bearing the single-site changes, His-177 → Gln or Tyr-345 → Cys, exhibited nearly identical binding properties to that of the full-length β-subunit. This is the first study in which regions of the isolated β-subunit of an ATP synthase complex have been systematically assessed by mutational analysis for their contribution to ATP binding. The results indicate that the A consensus sequence contributes over one-half of the total binding energy, with the C region making a significant contribution. The N-terminal region, the B region, Tyr-345, and His-177 appear to play little or no role in ATP binding.

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