Structural domain organization of gastric H+,K+-ATPase and its rearrangement during the catalytic cycle

Abstract

Differential scanning calorimetry has been used to characterized the thermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacity function of (H+,K+)-ATPase in highly oriented gastric vesicles displays two peaks at 53.9°C (T(m1)) and 61.8°C (T(m2)). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state processes. They can be assigned to two cooperative domains located in the cytoplasmic loops of the α-subunit, according to the disappearance of the endothermic signal upon removal of these regions by proteinase K digestion. Analysis of the thermal-induced unfolding of the enzyme trapped in different catalytic cycle intermediates has allowed us to get insight into the E1-E2 conformational change. In the E1 forms both transitions are always observed. As T(m1) is shifted to T(m2) by vanadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions. Stabilization of the E2 forms results in the disappearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in T(m2) and ΔH1. The catalytic domain melts following a process in which intermolecular interactions either in the native or in the unfolded state might be involved. Interestingly, the E2- vanadate-K+ form displays intermediate properties between the E1 and E2 conformational families.