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 ␣-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 E 1 -E 2 conformational change. In the E 1 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 E 2 forms results in the disappearance of the second transition at saturation by K ؉ , Mg 2؉ -ATP, and Mg 2؉-vanadate as well as in significant changes in T m2 and ⌬H 1 . 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 E 2 -vanadate-K ؉ form displays intermediate properties between the E 1 and E 2 conformational families.

The thermal unfolding and domain structure of Na+/K+‐exchanging ATPase.

“…However, we believe that the transmembrane fragments of the Na + /K + ‐ATPase are highly stable and do not contribute to the unfolding transition of this protein in the temperature range studied. Supporting this idea, no thermal transition has been observed in the proteolytically isolated membrane region of the H + /K + ‐ATPase homologous to the Na + /K + ‐ATPase [29].…”

“…Because much as the enthalpy of the third transition is dependent on ATP binding, one could suggest that it represents the large cytoplasmic loop. Remarkably, the cation occlusion and ATP binding sites have been assigned to two discrete folding domains of the α subunit in the homologous H + /K + ‐ATPase [29].…”

Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain

“…Gasset et al (30) observed two peaks of heat capacity at 53.9 and 61.8°C in gastric H,K-ATPase. We would have predicted that the heat transition at 53.9°C corresponds to the physical changes we observed at 55°C, but protection by K ϩ confounds this interpretation.…”

Structural difference in the H+,K+‐ATPase between the E1 and E2 conformations

“…Yet, no quantitative determination of the size of the protein region affected by the changes has been made. Recent differential scanning calorimetry experiments show that arrangement of two distinct structural domains characterized by two temperature‐induced cooperative unfolding are present in the E1 conformation (Na + buffer) but that one disappears when the enzyme is forced into the E2 conformation (K + buffer) [11]…”

“…One potential explanation for the vanishing transition in one of the protein domains is that this domain becomes embedded or partially embedded in the membrane [11]. The K + binding site has also been suggested to be on the H6 segment [12], a part of the H5‐H6 hairpin, accessible to trypsin cleavage in the absence of ligands but not in the presence of K + [13].…”