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

Abe K, Tani K, Fujiyoshi Y - Nat Commun (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-0852, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

SCH28080-bound E2P structure of H+,K+-ATPase analysed at 7 Å resolution.(a) Overall structure of the H+,K+-ATPase. The molecular surface represents the EM density map (contoured at 1σ) with a superimposed homology model of H+,K+-ATPase (ribbon). Bound SCH28080 at the luminal cavity and ADP at the N domain are shown as red and purple spheres, respectively. The yellow–brown box indicates the approximate location of the lipid bilayer. Colour code: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue (surface); β-subunit, red. Colour of the TM helices of the homology model gradually changes from M1 (blue) to M10 (red). As described in the text, the homology model does not contain the A-M2 linker (dotted line in blue). (b) SCH28080-binding site viewed from the luminal side (red arrow in a). The amino acids important for SCH28080 affinity are indicated as spheres (red, more than 10 times lower affinity; orange: 3–10 times lower, based on mutagenesis studies)202122232425262728 to show their locations on the chain trace of the homology model (ribbon, colour codes as in a). Green mesh represents the excess density (that is, [EM density map (1σ, white surface)]−[homology model], see text for details), showing good fit with the predicted position of SCH28080 by the docking simulation (stick model, see also Fig. 2a and Discussion). (c) A cross-section of the TM region perpendicular to the membrane plane. The EM density map contoured at 1σ is shown as a white surface, and the sectional surface is shown in blue. Dotted spheres indicate K+-binding sites of the Na+,K+-ATPase20 to show the approximate location of the cation binding site on the H+,K+-ATPase, yet these ions are not resolved in the present (SCH)E2BeF structure because of the limited resolution of the EM density map and K+-free conditions for the 2D crystallization. The M6 helix was omitted for clarity. Bound SCH28080 (red stick, position predicted by the docking simulation) is found at the centre of the luminal cavity, apparently blocking the K+-entry pathway from the luminal side of the membrane.
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f1: SCH28080-bound E2P structure of H+,K+-ATPase analysed at 7 Å resolution.(a) Overall structure of the H+,K+-ATPase. The molecular surface represents the EM density map (contoured at 1σ) with a superimposed homology model of H+,K+-ATPase (ribbon). Bound SCH28080 at the luminal cavity and ADP at the N domain are shown as red and purple spheres, respectively. The yellow–brown box indicates the approximate location of the lipid bilayer. Colour code: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue (surface); β-subunit, red. Colour of the TM helices of the homology model gradually changes from M1 (blue) to M10 (red). As described in the text, the homology model does not contain the A-M2 linker (dotted line in blue). (b) SCH28080-binding site viewed from the luminal side (red arrow in a). The amino acids important for SCH28080 affinity are indicated as spheres (red, more than 10 times lower affinity; orange: 3–10 times lower, based on mutagenesis studies)202122232425262728 to show their locations on the chain trace of the homology model (ribbon, colour codes as in a). Green mesh represents the excess density (that is, [EM density map (1σ, white surface)]−[homology model], see text for details), showing good fit with the predicted position of SCH28080 by the docking simulation (stick model, see also Fig. 2a and Discussion). (c) A cross-section of the TM region perpendicular to the membrane plane. The EM density map contoured at 1σ is shown as a white surface, and the sectional surface is shown in blue. Dotted spheres indicate K+-binding sites of the Na+,K+-ATPase20 to show the approximate location of the cation binding site on the H+,K+-ATPase, yet these ions are not resolved in the present (SCH)E2BeF structure because of the limited resolution of the EM density map and K+-free conditions for the 2D crystallization. The M6 helix was omitted for clarity. Bound SCH28080 (red stick, position predicted by the docking simulation) is found at the centre of the luminal cavity, apparently blocking the K+-entry pathway from the luminal side of the membrane.

Mentions: To elucidate the inhibitor-binding site and induced conformational changes of H+,K+-ATPase, we prepared two-dimensional (2D) crystals in the presence of the phosphate analogue BeF and SCH28080, a P-CAB prototype. Similar to the previously reported BeF-bound 2D crystals31, large tubular crystals were formed in the presence of SCH28080 (Supplementary Fig. S1a), and were analysed by electron crystallography using cryo-electron microscopy32 (Supplementary Fig. S1 and Supplementary Table S1). The final three-dimensional electron microscopic density map (EM density map) was calculated to 7 Å resolution (Fig. 1a, Supplementary Movie S1). Consistent with the crystallization conditions, the overall structure of the present EM density map (Fig. 1a) shows characteristic features of the ADP-insensitive E2P conformation to which SCH28080 is preferentially bound (Supplementary Fig. S2)16. The BeF-bound phosphorylation site (Asp385) at the P domain is covered by the A domain, and the ADP-bound N domain is separated from the P domain, thus the bound phosphate analogue seems to be isolated from both ADP and the bulk solution (Supplementary Fig. S3). The structure is not identical, however, to that of previously reported pseudo-E2P states1431 (as both the aluminium fluoride (AlF)-bound E2AlF and the BeF-bound E2BeF structures of H+,K+-ATPase are indistinguishable at the currently achieved resolution, they are abbreviated as E2P* unless otherwise stated). Therefore, the present structure is designated as an SCH28080-bound pseudo-E2P state, (SCH)E2BeF, which represents a conformation different from that of the inhibitor-free E2P* structures described later. Because of the similarity in their primary sequences, overall conformations, and representative reaction states (inhibitor-bound E2P state), we used the ouabain-bound Na+,K+-ATPase20 atomic model (PDB ID: 3A3Y) as a starting template for the homology model. The EM density map allowed us to determine the position of the individual helices in the TM domain, as well as in the cytoplasmic domains, and we successfully generated a homology model of H+,K+-ATPase by fitting them into the EM density map as rigid bodies.


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

Abe K, Tani K, Fujiyoshi Y - Nat Commun (2011)

SCH28080-bound E2P structure of H+,K+-ATPase analysed at 7 Å resolution.(a) Overall structure of the H+,K+-ATPase. The molecular surface represents the EM density map (contoured at 1σ) with a superimposed homology model of H+,K+-ATPase (ribbon). Bound SCH28080 at the luminal cavity and ADP at the N domain are shown as red and purple spheres, respectively. The yellow–brown box indicates the approximate location of the lipid bilayer. Colour code: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue (surface); β-subunit, red. Colour of the TM helices of the homology model gradually changes from M1 (blue) to M10 (red). As described in the text, the homology model does not contain the A-M2 linker (dotted line in blue). (b) SCH28080-binding site viewed from the luminal side (red arrow in a). The amino acids important for SCH28080 affinity are indicated as spheres (red, more than 10 times lower affinity; orange: 3–10 times lower, based on mutagenesis studies)202122232425262728 to show their locations on the chain trace of the homology model (ribbon, colour codes as in a). Green mesh represents the excess density (that is, [EM density map (1σ, white surface)]−[homology model], see text for details), showing good fit with the predicted position of SCH28080 by the docking simulation (stick model, see also Fig. 2a and Discussion). (c) A cross-section of the TM region perpendicular to the membrane plane. The EM density map contoured at 1σ is shown as a white surface, and the sectional surface is shown in blue. Dotted spheres indicate K+-binding sites of the Na+,K+-ATPase20 to show the approximate location of the cation binding site on the H+,K+-ATPase, yet these ions are not resolved in the present (SCH)E2BeF structure because of the limited resolution of the EM density map and K+-free conditions for the 2D crystallization. The M6 helix was omitted for clarity. Bound SCH28080 (red stick, position predicted by the docking simulation) is found at the centre of the luminal cavity, apparently blocking the K+-entry pathway from the luminal side of the membrane.
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f1: SCH28080-bound E2P structure of H+,K+-ATPase analysed at 7 Å resolution.(a) Overall structure of the H+,K+-ATPase. The molecular surface represents the EM density map (contoured at 1σ) with a superimposed homology model of H+,K+-ATPase (ribbon). Bound SCH28080 at the luminal cavity and ADP at the N domain are shown as red and purple spheres, respectively. The yellow–brown box indicates the approximate location of the lipid bilayer. Colour code: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue (surface); β-subunit, red. Colour of the TM helices of the homology model gradually changes from M1 (blue) to M10 (red). As described in the text, the homology model does not contain the A-M2 linker (dotted line in blue). (b) SCH28080-binding site viewed from the luminal side (red arrow in a). The amino acids important for SCH28080 affinity are indicated as spheres (red, more than 10 times lower affinity; orange: 3–10 times lower, based on mutagenesis studies)202122232425262728 to show their locations on the chain trace of the homology model (ribbon, colour codes as in a). Green mesh represents the excess density (that is, [EM density map (1σ, white surface)]−[homology model], see text for details), showing good fit with the predicted position of SCH28080 by the docking simulation (stick model, see also Fig. 2a and Discussion). (c) A cross-section of the TM region perpendicular to the membrane plane. The EM density map contoured at 1σ is shown as a white surface, and the sectional surface is shown in blue. Dotted spheres indicate K+-binding sites of the Na+,K+-ATPase20 to show the approximate location of the cation binding site on the H+,K+-ATPase, yet these ions are not resolved in the present (SCH)E2BeF structure because of the limited resolution of the EM density map and K+-free conditions for the 2D crystallization. The M6 helix was omitted for clarity. Bound SCH28080 (red stick, position predicted by the docking simulation) is found at the centre of the luminal cavity, apparently blocking the K+-entry pathway from the luminal side of the membrane.
Mentions: To elucidate the inhibitor-binding site and induced conformational changes of H+,K+-ATPase, we prepared two-dimensional (2D) crystals in the presence of the phosphate analogue BeF and SCH28080, a P-CAB prototype. Similar to the previously reported BeF-bound 2D crystals31, large tubular crystals were formed in the presence of SCH28080 (Supplementary Fig. S1a), and were analysed by electron crystallography using cryo-electron microscopy32 (Supplementary Fig. S1 and Supplementary Table S1). The final three-dimensional electron microscopic density map (EM density map) was calculated to 7 Å resolution (Fig. 1a, Supplementary Movie S1). Consistent with the crystallization conditions, the overall structure of the present EM density map (Fig. 1a) shows characteristic features of the ADP-insensitive E2P conformation to which SCH28080 is preferentially bound (Supplementary Fig. S2)16. The BeF-bound phosphorylation site (Asp385) at the P domain is covered by the A domain, and the ADP-bound N domain is separated from the P domain, thus the bound phosphate analogue seems to be isolated from both ADP and the bulk solution (Supplementary Fig. S3). The structure is not identical, however, to that of previously reported pseudo-E2P states1431 (as both the aluminium fluoride (AlF)-bound E2AlF and the BeF-bound E2BeF structures of H+,K+-ATPase are indistinguishable at the currently achieved resolution, they are abbreviated as E2P* unless otherwise stated). Therefore, the present structure is designated as an SCH28080-bound pseudo-E2P state, (SCH)E2BeF, which represents a conformation different from that of the inhibitor-free E2P* structures described later. Because of the similarity in their primary sequences, overall conformations, and representative reaction states (inhibitor-bound E2P state), we used the ouabain-bound Na+,K+-ATPase20 atomic model (PDB ID: 3A3Y) as a starting template for the homology model. The EM density map allowed us to determine the position of the individual helices in the TM domain, as well as in the cytoplasmic domains, and we successfully generated a homology model of H+,K+-ATPase by fitting them into the EM density map as rigid bodies.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-0852, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus