An ion gating mechanism of gastric H,K-ATPase based on molecular dynamics simulations.
Gastric H,K-ATPase is an electroneutral transmembrane pump that moves protons from the cytoplasm of the parietal cell into the gastric lumen in exchange for potassium ions. The mechanism of transport against the established electrochemical gradients includes intermediate conformations in which the transferred ions are trapped (occluded) within the membrane domain of the pump. The pump cycle involves switching between the E1 and E2P states. Molecular dynamics simulations on homology models of the E2P and E1 states were performed to investigate the mechanism of K(+) movement in this enzyme. We performed separate E2P simulations with one K(+) in the luminal channel, one K(+) ion in the occlusion site, two K(+) ions in the occlusion site, and targeted molecular dynamics from E2P to E1 with two K(+) ions in the occlusion site. The models were inserted into a lipid bilayer system and were stable over the time course of the simulations, and K(+) ions in the channel moved to a consistent location near the center of the membrane domain, thus defining the occlusion site. The backbone carbonyl oxygen from residues 337 through 342 on the nonhelical turn of M4, as well as side-chain oxygen from E343, E795, and E820, participated in the ion occlusion. A single water molecule was stably bound between the two K(+) ions in the occlusion site, providing an additional ligand and partial shielding the positive charges from one another. Targeted molecular dynamics was used to transform the protein from the E2P to the E1 state (two K(+) ions to the cytoplasm). This simulation identified the separation of the water column in the entry channel as the likely gating mechanism on the luminal side. A hydrated exit channel also formed on the cytoplasmic side of the occlusion site during this simulation. Hence, water molecules became available to hydrate the ions. The movement of the M1M2 transmembrane segments, and the displacement of residues Q159, E160, Q110, and T152 during the conformational change, as well as the motions of E343 and L346, acted as the cytoplasmic-side gate.
Lawrence Livermore National Laboratory, Livermore, California 94550, USA.
Richard J
RJ
Law
Keith
K
Munson
George
G
Sachs
Felice C
FC
Lightstone
D003593:Cytoplasm / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D015640:Ion Channel Gating; D008958:Models, Molecular; D009068:Movement; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D013270:Stomach / Q000201:enzymology*; D014867:Water / Q000378:metabolism
D014867:Water; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium