Abstract
Gastric H(+),K(+)-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K(+)-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H(+),K(+)-ATPase is consistent with that observed in sarcoplasmic reticulum Ca(2+)-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.
Highlights
The gastric Hϩ,Kϩ-ATPase proton pump achieves gastric acid secretion
Two-dimensional Crystallization of (SCH)E21⁄7AlF and (SCH)E21⁄7 magnesium fluoride (MgF) States—Previously, we reported several cryo-electron microscopy (EM) structures of Hϩ,Kϩ-ATPase bound to different XFs in the presence or absence of ions and substrates for the TM domain, which include the reaction state analog of E21⁄7BeF [17], E21⁄7AlF [13], (SCH)E21⁄7BeF [18], and (Rbϩ)E21⁄7AlF [19]
Because inhibition of Hϩ,Kϩ-ATPase activity by MgF is reversible and weaker than that of other XFs [17], stabilization of the molecular conformation by SCH28080 is required for the twodimensional crystal formation when MgF is used as the phosphate analog
Summary
The gastric Hϩ,Kϩ-ATPase proton pump achieves gastric acid secretion. Results: A newly determined (SCH)E21⁄7MgF structure represents a hybrid conformation of (SCH)E21⁄7BeF and luminal-closed (Rbϩ)E21⁄7AlF states. The molecular conformation of the (SCH)E21⁄7MgF state represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. To clarify the transport mechanism of Hϩ,Kϩ-ATPase, a better understanding of the E2P state structure as a key reaction intermediate is essential to reveal the conformational changes that occur for the Hϩ/Kϩ exchange against a luminal solution with opening and closing of the transport pathway and gate. Even at a limited resolution of ϳ8 Å determined by electron crystallography, molecular events occurring at the phosphorylation site and the luminal gate can be observed based on the rearrangement of the cytoplasmic domains and TM helices, respectively, in response to each bound XF [13, 17,18,19]. Systematic comparison of the cryo-EM structures in the E2P-related states allows us to propose a luminal gating mechanism of Hϩ,KϩATPase
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