title: Mutational analysis of the consensus nucleotide binding sequences in the rat liver mitochondrial ATP synthase beta-subunit.
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 beta-subunit of the ATP synthase complex. A molecular description of the ATP binding site on the beta-subunit is, therefore, critical to understanding the mechanism of coupling in the enzyme. Previously, we reported that a purified, bacterially expressed rat liver beta-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 beta-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 overexpressed full-length beta-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 beta-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.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: P J
authors.initials: PJ
authors.lastname: Thomas
authors.forename: D N
authors.initials: DN
authors.lastname: Garboczi
authors.forename: P L
authors.initials: PL
authors.lastname: Pedersen
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: D000227:Adenine Nucleotides; D006180:Proton-Translocating ATPases