The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli.
The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches. First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function. This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis. Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122. Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected. Double mutants were constructed with a mutation that permits cross-linking to the b subunit. Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114. Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA. In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation.
D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D017136:Ion Transport; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D025243:Bacterial Proton-Translocating ATPases; D025261:Mitochondrial Proton-Translocating ATPases; C074428:atpB protein, E coli