Structural analysis of 2D crystals of gastric H+,K+-ATPase in different states of the transport cycle.
The H+,K+-ATPase uses ATP to pump protons across the gastric membrane. We used electron crystallography and limited trypsin proteolysis to study conformational changes in the H+,K+-ATPase. Well-ordered 2D crystals were obtained with detergent-solubilized H+,K+-ATPase at low pH in the absence of nucleotides, E1 state, and in the presence of fluoroaluminate and ADP, mimicking the E1PADP state. Projection maps obtained with frozen-hydrated two-dimensional crystals of the H+,K+-ATPase in these two states looked very similar, suggesting only small conformational changes during the transition from the E1 to the E1P x ADP state. This result differs from the X-ray crystal structures of the related ATPase SERCA, which revealed substantially different conformations in the E1 and E1P x ADP states. To further characterize the conformational changes in the H+,K+-ATPase during its transport cycle, we performed limited proteolysis with trypsin. All examined states of the H+,K+-ATPase, including the E1 and E1P x ADP states present in the 2D crystals,showed characteristic differences in the digestion patterns. While the results from the limited proteolysis experiments thus show that the H+,K+-ATPase adopts distinct conformations during different stages of the transport cycle, the projection maps indicate that the structural rearrangements in the H+,K+-ATPase are much smaller than those observed in the related SERCA ATPase.
Nishizawa
Tomohiro
T
Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.
Abe
Kazuhiro
K
Tani
Kazutoshi
K
Fujiyoshi
Yoshinori
Y
D000244:Adenosine Diphosphate; D000818:Animals; D001693:Biological Transport, Active; D020285:Cryoelectron Microscopy; D003460:Crystallization; D006863:Hydrogen-Ion Concentration; D044367:Phase Transition; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013270:Stomach / Q000201:enzymology; D013552:Swine; D014867:Water
D014867:Water; D000244:Adenosine Diphosphate; D000254:Sodium-Potassium-Exchanging ATPase