Kazuhiro Abe

Inter‐subunit interaction of gastric H+,K+‐ATPase prevents reverse reaction of the transport cycle

The gastric H+,K+‐ATPase is an ATP‐driven proton pump responsible for generating a million‐fold proton gradient across the gastric membrane. We present the structure of gastric H+,K+‐ATPase at 6.5 Å resolution as determined by electron crystallography of two‐dimensional crystals. The structure shows the catalytic α‐subunit and the non‐catalytic β‐subunit in a pseudo‐E2P conformation. Different from Na+,K+‐ATPase, the N‐terminal tail of the β‐subunit is in direct contact with the phosphorylation domain of the α‐subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β‐subunit N‐terminus allowed the reverse reaction to occur. These results suggest that the β‐subunit N‐terminus prevents the reverse reaction from E2P to E1P, which is likely to be relevant for the generation of a large H+ gradient in vivo situation.

Conformational rearrangement of gastric H(+),K(+)-ATPase induced by an acid suppressant.

Acid-related gastric diseases are associated with disorder of digestive tract acidification. The gastric proton pump, H+,K+-ATPase, exports H+ in exchange for luminal K+ to generate a highly acidic environment in the stomach, and is a main target for acid suppressants. Here, we report the three-dimensional structure of gastric H+,K+-ATPase with bound SCH28080, a representative K+-competitive acid blocker, at 7 Å resolution based on electron crystallography of two-dimensional crystals. The density of the bound SCH28080 is found near transmembrane (TM) helices 4, 5 and 6, in the luminal cavity. The SCH28080-binding site is formed by the rearrangement of TM helices, which is in turn transmitted to the cytoplasmic domains, resulting in a luminal-open conformation. These results represent the first structural evidence for a binding site of an acid suppressant on H+,K+-ATPase, and the conformational change induced by this class of drugs.

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

Gastric H+,K+-ATPase is responsible for gastric acid secretion. ATP-driven 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 86Rb+ 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 106-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.

E2P-state stabilization by the N-terminal tail of the H,K-ATPase β-subunit is critical for efficient proton pumping under in vivo conditions

The catalytic α-subunits of Na,K- and H,K-ATPase require an accessory β-subunit for proper folding, maturation, and plasma membrane delivery but also for cation transport. To investigate the functional significance of the β-N terminus of the gastric H,K-ATPase in vivo, several N-terminally truncated β-variants were expressed in Xenopus oocytes, together with the S806C α-subunit variant. Upon labeling with the reporter fluorophore tetramethylrho da mine-6-maleimide, this construct can be used to determine the voltage-dependent distribution between E1P/E2P states. Whereas the E1P/E2P conformational equilibrium was unaffected for the shorter N-terminal deletions βΔ4 and βΔ8, we observed significant shifts toward E1P for the two larger deletions βΔ13 and βΔ29. Moreover, the reduced ΔF/F ratios of βΔ13 and βΔ29 indicated an increased reverse reaction via E2P → E1P + ADP → E1 + ATP, because cell surface expression was completely unaffected. This interpretation is supported by the reduced sensitivity of the mutants toward the E2P-specific inhibitor SCH28080, which becomes especially apparent at high concentrations (100 μm). Despite unaltered apparent Rb+ affinities, the maximal Rb+ uptake of these mutants was also significantly lowered. Considering the two putative interaction sites between the β-N terminus and α-subunit revealed by the recent cryo-EM structure, the N-terminal tail of the H,K-ATPase β-subunit may stabilize the pump in the E2P conformation, thereby increasing the efficiency of proton release against the million-fold proton gradient of the stomach lumen. Finally, we demonstrate that a similar truncation of the β-N terminus of the closely related Na,K-ATPase does not affect the E1P/E2P distribution or pump activity, indicating that the E2P-stabilizing effect by the β-N terminus is apparently a unique property of the H,K-ATPase.

The four-transmembrane protein IP39 of Euglena forms strands by a trimeric unit repeat

Euglenoid flagellates have striped surface structures comprising pellicles, which allow the cell shape to vary from rigid to flexible during the characteristic movement of the flagellates. In Euglena gracilis, the pellicular strip membranes are covered with paracrystalline arrays of a major integral membrane protein, IP39, a putative four-membrane-spanning protein with the conserved sequence motif of the PMP-22/EMP/MP20/Claudin superfamily. Here we report the three-dimensional structure of Euglena IP39 determined by electron crystallography. Two-dimensional crystals of IP39 appear to form a striated pattern of antiparallel double-rows in which trimeric IP39 units are longitudinally polymerised, resulting in continuously extending zigzag-shaped lines. Structural analysis revealed an asymmetric molecular arrangement in the trimer, and suggested that at least four different interactions between neighbouring protomers are involved. A combination of such multiple interactions would be important for linear strand formation of membrane proteins in a lipid bilayer.

Structural and functional characterization of H+,K+-ATPase with bound fluorinated phosphate analogs

Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. In order to characterize the phosphorylation events on H(+),K(+)-ATPase, the properties of fluorinated phosphate analogs [XFs, e.g. aluminum fluoride (AlF), beryllium fluoride (BeF) and magnesium fluoride (MgF)], and the structural differences induced by XFs were investigated. The addition of divalent cations to the XF-inhibited H(+),K(+)-ATPase restores the activity of the AlF- or MgF-inhibited, but not of the BeF-inhibited enzyme, although limited trypsin digestion reveals that they assume the same E(2)P-like state. To clarify the conformational differences induced by XFs, the structure of BeF-bound H(+),K(+)-ATPase was analyzed at 8A resolution. The structure is almost identical to the previously reported AlF-bound E(2)P structure, unlike the distinctive X-ray structure of BeF-bound SERCA, in which the luminal gate was observed to be widely opened. Since the analyzed structure of the H(+),K(+)-ATPase revealed that both AlF and BeF-bound to the P domain were not exposed to the solvent, the dissociation of XFs induced by divalent cations could be interpreted in terms of stability against thermal fluctuations. Furthermore, the conformational differences found between the cytoplasmic domains of H(+),K(+)-ATPase and SERCA provide a framework to understand the characteristic mechanism, by which divalent cations reactivate the XF-inhibited H(+),K(+)-ATPase.

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.

Carbon sandwich preparation preserves quality of two-dimensional crystals for cryo-electron microscopy

Received 7 June 2013; accepted 21 June 2013Abstract Electron crystallography is an important method for determining the structure of membrane proteins. In this paper, we show the impact of a carbon sandwich preparation on the preservation of crystalline sample quality, using characteristic examples of two-dimensional (2D) crystals from gastric H+,K+-ATPase and their analyzed images. Compared with the ordinary single carbon support film preparation, the carbon sandwich preparation dramatically enhanced the resolution of images from flat sheet 2D crystals. As water evaporation is restricted in the carbon-sandwiched specimen, the improvement could be due to the strong protective effect of the retained water against drastic changes in the environment surrounding the specimen, such as dehydration and increased salt concentrations. This protective effect by the carbon sandwich technique helped to maintain the inherent and therefore best crystal conditions for analysis. Together with its strong compensation effect for the image shift due to beam-induced specimen charging, the carbon sandwich technique is a powerful method for preserving crystals of membrane proteins with larger hydrophilic regions, such as H+,K+-ATPase, and thus constitutes an efficient and high-quality method for collecting data for the structural analysis of these types of membrane proteins by electron crystallography.

K+ binding and proton redistribution in the E2P state of the H+, K+-ATPase

The H+, K+-ATPase (HKA) uses ATP to pump protons into the gastric lumen against a million-fold proton concentration gradient while counter-transporting K+ from the lumen. The mechanism of release of a proton into a highly acidic stomach environment, and the subsequent binding of a K+ ion necessitates a network of protonable residues and dynamically changing protonation states in the cation binding pocket dominated by five acidic amino acid residues E343, E795, E820, D824, and D942. We perform molecular dynamics simulations of spontaneous K+ binding to all possible protonation combinations of the acidic amino acids and carry out free energy calculations to determine the optimal protonation state of the luminal-open E2P state of the pump which is ready to bind luminal K+. A dynamic pKa correlation analysis reveals the likelihood of proton transfer events within the cation binding pocket. In agreement with in-vitro measurements, we find that E795 is likely to be protonated, and that E820 is at the center of the proton transfer network in the luminal-open E2P state. The acidic residues D942 and D824 are likely to remain protonated, and the proton redistribution occurs predominantly amongst the glutamate residues exposed to the lumen. The analysis also shows that a lower number of K+ ions bind at lower pH, modeled by a higher number of protons in the cation binding pocket, in agreement with the ‘transport stoichiometry variation’ hypothesis.

Systematic Comparison of Molecular Conformations of H+,K+-ATPase Reveals an Important Contribution of the A-M2 Linker for the Luminal Gating

Background: The gastric H+,K+-ATPase proton pump achieves gastric acid secretion. Results: A newly determined (SCH)E2·MgF structure represents a hybrid conformation of (SCH)E2·BeF and luminal-closed (Rb+)E2·AlF states. Conclusion: Comparison of E2P-related structures revealed that the A-M2 linker importantly contributes to conformational changes of the enzyme. Significance: A-M2 linker-mediated conformational change is conserved between H+,K+-ATPase and Ca2+-ATPase and perhaps other P-type ATPases. 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 Ca2+-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.