The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain undergoes drastic conformational changes between a non-inhibited form (down-state) and an inhibited form (up-state). Even though this C-terminal domain does not appear to be essential for ATP synthesis activity, there are evidence of its involvement in the coupling mechanism of the pump [1]. Recently, it has been proposed that the coupling degree of the ATP-synthase can vary as a function of ADP and Pi concentration [2-4]. In the present work we explored the possible role of the C-terminal domain in this ligand-dependent uncoupling, by examining a C-terminally truncated e mutant of E.coli. We have developed a low copy number expression vector carrying an extra copy of uncC with the aim of promoting normal levels of assembly of the mutated ATP-synthase complex. Both the wild-type and the ε88-stop truncated strains showed well energized membranes. Noticeably, they showed a marked difference in their response to Pi: the Pi-induced inhibition of membrane-bound ATPase activity appeared to be completely lost in the truncated mutant, and the Pi-induced coupling increase was very reduced. However, pre-incubation of the mutated enzyme with ADP at rather low concentrations ([ADP] = 100 nM) largely restored the Pi-induced hydrolysis inhibition. Analogously, the increase in coupling degree induced by Pi was resumed after incubation with extremely low [ADP] (1 nM). This suggests that, contrary to wild-type, the truncated mutant had lost its bound ADP, most likely during membrane preparation, as a consequence of a lower affinity for ADP. The whole set of data is interpreted to indicate that, in the wild-type ATP-synthase, one ADP-binding site at very high affinity (Kd < 1 nM) mainly influences the coupling degree, and one ADP-binding site atintermediate affinity mainly inhibits the hydrolytic activity. The ε-subunit C-terminal domain appears to increase the affinity of these two ADP binding sites, thus playing a major role in modulating both the activity and coupling degree of the ATP-synthase.

Role of the epsilon subunit C-terminal domain of the Escherichia coli ATP synthase in modulating activity and coupling degree

D'ALESSANDRO, MANUELA;TURINA, MARIA PAOLA;MELANDRI, BRUNO ANDREA;
2012

Abstract

The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain undergoes drastic conformational changes between a non-inhibited form (down-state) and an inhibited form (up-state). Even though this C-terminal domain does not appear to be essential for ATP synthesis activity, there are evidence of its involvement in the coupling mechanism of the pump [1]. Recently, it has been proposed that the coupling degree of the ATP-synthase can vary as a function of ADP and Pi concentration [2-4]. In the present work we explored the possible role of the C-terminal domain in this ligand-dependent uncoupling, by examining a C-terminally truncated e mutant of E.coli. We have developed a low copy number expression vector carrying an extra copy of uncC with the aim of promoting normal levels of assembly of the mutated ATP-synthase complex. Both the wild-type and the ε88-stop truncated strains showed well energized membranes. Noticeably, they showed a marked difference in their response to Pi: the Pi-induced inhibition of membrane-bound ATPase activity appeared to be completely lost in the truncated mutant, and the Pi-induced coupling increase was very reduced. However, pre-incubation of the mutated enzyme with ADP at rather low concentrations ([ADP] = 100 nM) largely restored the Pi-induced hydrolysis inhibition. Analogously, the increase in coupling degree induced by Pi was resumed after incubation with extremely low [ADP] (1 nM). This suggests that, contrary to wild-type, the truncated mutant had lost its bound ADP, most likely during membrane preparation, as a consequence of a lower affinity for ADP. The whole set of data is interpreted to indicate that, in the wild-type ATP-synthase, one ADP-binding site at very high affinity (Kd < 1 nM) mainly influences the coupling degree, and one ADP-binding site atintermediate affinity mainly inhibits the hydrolytic activity. The ε-subunit C-terminal domain appears to increase the affinity of these two ADP binding sites, thus playing a major role in modulating both the activity and coupling degree of the ATP-synthase.
Biochimica et Biophysica Acta (BBA) - Bioenergetics
S10
S10
M. D'Alessandro;P. Turina;B.A. Melandri;S.D. Dunn
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/152597
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