Mitochondrial F-type ATPases: the glycine-rich loop of the beta-subunit is a pyrophosphate binding domain.
The beta-subunit of the mitochondrial ATP synthase complex comprises the bulk, if not all, of the catalytic nucleotide binding site on the enzyme. A region of homologous sequence rich in glycines (G) and containing a basic lysine (K) and a threonine (T) is found in the beta-subunit as well as many other purine nucleotide binding proteins. The consensus sequence of this region is Gx4GKT, where x represents any amino acid, and is called the A region or glycine-rich loop. The related function of these proteins implies that the glycine-rich loop is directly involved in nucleotide binding. Here we directly test the involvement of the beta-subunit's glycine-rich region in adenine nucleotide binding using two independent approaches. A synthetic fifty amino acid peptide, PP-50, containing the glycine-rich region and the surrounding sequence was assessed for secondary structure and interaction with potential ligands. Circular dichroism spectropolarimetry indicates that PP-50 assumes a predominantly beta-sheet conformation in solution. Significantly, the peptide precipitates from solution when ATP, ADP, GTP, ITP, and pyrophosphate are added, but not when AMP or phosphate are included. Magnesium is not required for the interaction with the purine nucleotides. Complimentary to these studies, the sequence around the Gx4GKT motif was deleted from a recombinant rat liver beta-subunit overexpressed in E. coli. While the wild type beta-subunit showed specificity for the tri- and diphosphonucleotides, the deletion mutant bound tri-, di-, and monophosphate nucleotides with equal affinity.(ABSTRACT TRUNCATED AT 250 WORDS)
Thomas
P J
PJ
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Garboczi
D N
DN
Pedersen
P L
PL
D000227:Adenine Nucleotides / Q000378:metabolism; D000595:Amino Acid Sequence; D011756:Diphosphates / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D008024:Ligands; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011685:Purine Nucleotides / Q000378:metabolism
D000227:Adenine Nucleotides; D011756:Diphosphates; D008024:Ligands; D011685:Purine Nucleotides; D006180:Proton-Translocating ATPases; D005998:Glycine