Cryo-EM structure of gastric H ⁺ ,K ⁺ -ATPase with a single occupied cation-binding site

Abstract

Gastric H + ,K + -ATPase is responsible for gastric acid secretion. ATPdriven H + uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K + , resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H + /2K + to 1H + /1K + per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K + congener (Rb + ), the 8-Å resolution structure of H + ,K + -ATPase in the transition state of dephosphorylation, (Rb + )E2∼AlF, which is distinct from the preceding Rb + -free E2P state. A strong density located in the transmembrane cation-binding site of (Rb + )E2∼AlF highly likely represents a single bound Rb + ion, which is clearly different from the Rb + -free E2AlF or K + -bound (K + )E2∼AlF structures. Measurement of radioactive 86 Rb + binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb + is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H + /1K + /1ATP transport mode of H + ,K + -ATPase, which is a prerequisite for generation of the 10 6 -fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the β subunit's N terminus and the P domain observed in the (Rb + )E2∼AlF structure, we propose a refined vectorial transport model of H + ,K + -ATPase, which must prevail against the highly acidic state of the gastric lumen.electron crystallography | P-type ATPases | membrane proteins | bioenergetics L ike other P-type ATPases (1), the vectorial cation transport of gastric H + ,K + -ATPase (2) is accomplished by cyclical conformational changes of the enzyme (abbreviated as E), generally described using an E1/E2 nomenclature based on the PostAlbers scheme for Na + ,K + -ATPase (3) (SI Appendix, Fig. S1). In contrast to the closely related Na + ,K + -ATPase, which exchanges three Na + for two K + ions in an electrogenic transport reaction, gastric H + ,K + -ATPase operates electroneutrally, although its transport stoichiometry (2H + /2K + /ATP or 1H + /1K + /ATP) has remained controversial (4, 5). A measurement of the proton transport (5) revealed an H + /ATP ratio of 2 at neutral pH. Accordingly, two K + ions must be counter transported during a single turnover of the transport cycle, accompanied by the hydrolysis of one ATP molecule (2H + /2K + /ATP). The reported free energy for ATP hydrolysis of the gastric secretory membrane [−13 kcal/mol (4)] provides sufficient energy to achieve a maximum change in pH (ΔpH) of 4.7 units when two H + ions are transported per hydrolysis of one ATP molecule; thus, the generation of pH 1 (ΔpH > 6 units in the stomach) with a 2H + /2K + / ATP stoichiometry is thermodynamically impossible. Therefore,...

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

“…A low-resolution crystal structure of the H + ,K + -ATPase obtained at acidic pH in the presence of the K + congener Rb + shows that one bound Rb + ion occupies a site equivalent to site II of the Na + ,K + -ATPase, whereas the occupancy of site I is very low (8).…”

Human Type IV P-type ATPases That Work as Plasma Membrane Phospholipid Flippases and Their Regulation by Caspase and Calcium

“…ATPase Assay-The ATPase activity of P4-ATPase was determined as described (27)(28)(29)(30)(31) with slight modifications. In brief, the purified protein (10 ng) was preincubated for 10 min at room temperature in 40 l of the reaction mixture (40 mM MES/Tris buffer (pH 7.0), 150 mM NaCl, 5% glycerol, 5 mM MgCl 2 , 7 mM DTT, 600 M ATP, and 0.05% LMNG) and incubated for 20 min at 37°C.…”

P2C-Type ATPases and Their Regulation

“…It has been reported that Na + /K + -ATPase and SERCA sequence structures are 30 % identical and 65 % similar [2], this is the reason why SERCA model was used for Na + /K + -ATPase studies until it was crystalized. Also, both proteins have an α subunit, with 10 transmembrane spans, but Na + /K + -ATPase differs in having also β and γ isoforms, with 12 transmembrane spans, so two subunits should affect the α subunit conformation of Na + /K + -ATPase [3,4]. In this review, we concentrate only in P2C-ATPases: H + /K + -ATPases and Na + /K + -ATPase.…”