F 1-ATPase α-subunit made up from two fragments (1–395, 396–503) is stabilized by ATP and complexes containing it obey altered kinetics

Abstract

Inferred from the crystal structure of mitochondrial F 1-ATPase (Abrahams, J.P. et al. (1994) Nature 370, 621–628), the proteinase-sensitive region around Phe-395 of thermophilic F 1-ATPase α-subunit corresponds to the loop which connects main part of the carboxyl-terminal helical bundle domain with the ATP binding domain. This loop is in contact with the γ- and adjacent β-subunits. Two polypeptides corresponding to the sequence 1–395 and 396–503 of the α-subunit were expressed in Escherichia coli cells and they were copurified as an apparently functional α-subunit (α(695/396)) made up of two polypeptides. The isolated α(395/396) was stabilized by ATP-Mg, but not by ADP-Mg, although it bound both ATP-Mg and ADP-Mg with similar affinities ( K d, 11 μM and 14 μM, respectively). The α(395/396) was reconstitutable into α(395/396) 3 β 3 and α(395/396) 3 β 3 γ complexes. Different from the intact α 3 β 3 complex, the α(395/396) 3 β 3 complex was significantly resistant to the ATP-Mg-induced dissociation into a α 1 β 1 heterodimers. ATP hydrolysis by the α(395/396) 3 β 3 γ complex underwent a slow initial phase, whereas the intact α 3 β 3 γ complex exhibited an accelerated initial phase. Steady-state ATPase activity at various ATP concentrations showed negative cooperativity for the intact α 3 β 3 γ complex but apparently positive cooperativity for the α(395/396) 3 β 3 γ complex. The ATPase activities at a saturating ATP concentration of the complexes containing the α(395/396) were 180% of those containing intact α-subunits. These results indicate that a loop around Phe-395 is involved in intersubunit interaction in F 1-ATPase.