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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-induced conformational changes at the cytoplasmic part of the H+,K+-ATPase.(a–c) Close-up view of the A-M2 linker in E2AlF (a), (SCH)E2BeF (b) and their comparison (c). Each surface (white for E2AlF, blue for (SCH)E2BeF) and ribbon model (grey for E2AlF, colour for (SCH)E2BeF is as in Fig. 1a) represents the EM density map and homology model of the corresponding structures, respectively. The magenta sphere indicates Q159, which on mutation to Asn showed fourfold lower affinity for SCH28080. The A-M2 linker of E2AlF or (SCH)E2BeF is indicated by orange or blue arrows, respectively, yet the (SCH)E2BeF homology model does not contain this part (dotted blue line in b, c, see also text). Note that density for the N-terminal domain of the α-subunit (αNt) is absent in the (SCH)E2BeF structure (b, dotted circle) in contrast to the E2AlF structure (a). (d) Conformational change of the cytoplasmic domains. The surface shows the EM density map of (SCH)E2BeF with its homology model superimposed (colour codes as in Fig. 1a: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue). Orange mesh represents the E2AlF EM density map. It should be noted that the inter-subunit interaction between the P domain and the N-terminal tail of the β-subunit (βNt) observed in the E2AlF structure is not present in the (SCH)E2BeF structure (arrowhead, see text for details). All the EM density maps are drawn with a 1σ contour level. White arrows in all panels indicate observed conformational changes during the E2AlF→(SCH)E2BeF transition.
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f4: SCH28080-induced conformational changes at the cytoplasmic part of the H+,K+-ATPase.(a–c) Close-up view of the A-M2 linker in E2AlF (a), (SCH)E2BeF (b) and their comparison (c). Each surface (white for E2AlF, blue for (SCH)E2BeF) and ribbon model (grey for E2AlF, colour for (SCH)E2BeF is as in Fig. 1a) represents the EM density map and homology model of the corresponding structures, respectively. The magenta sphere indicates Q159, which on mutation to Asn showed fourfold lower affinity for SCH28080. The A-M2 linker of E2AlF or (SCH)E2BeF is indicated by orange or blue arrows, respectively, yet the (SCH)E2BeF homology model does not contain this part (dotted blue line in b, c, see also text). Note that density for the N-terminal domain of the α-subunit (αNt) is absent in the (SCH)E2BeF structure (b, dotted circle) in contrast to the E2AlF structure (a). (d) Conformational change of the cytoplasmic domains. The surface shows the EM density map of (SCH)E2BeF with its homology model superimposed (colour codes as in Fig. 1a: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue). Orange mesh represents the E2AlF EM density map. It should be noted that the inter-subunit interaction between the P domain and the N-terminal tail of the β-subunit (βNt) observed in the E2AlF structure is not present in the (SCH)E2BeF structure (arrowhead, see text for details). All the EM density maps are drawn with a 1σ contour level. White arrows in all panels indicate observed conformational changes during the E2AlF→(SCH)E2BeF transition.

Mentions: The conformational change observed in the TM region is transmitted to the cytoplasmic part of the enzyme, mostly by connecting linker between the A domain and M2 (A-M2 linker, Fig. 4a–c). Because of the observed movement of the M1–M2 bundle on SCH28080 binding (Fig. 3b), the A-M2 linker forms an upright rod-like conformation separate from the P domain in the (SCH)E2BeF structure (Fig. 4b,c), which is in distinct contrast to its configuration in E2P* (Fig. 4a)1431. It should be noted that the A-M2 linker is missing in this (SCH)E2BeF homology model (Fig. 4b,c, dashed line, F161–P175), because of the large difference between the EM density map and the starting template structure (ouabain-bound Na+,K+-ATPase20). Comparison of the A-M2 linker conformations among several known structures of other P2-type ATPases202130, however, reveals that it can assume mainly two conformations (Supplementary Fig. S6). The A-M2 linker is unwound in structures of the E2AlF conformation of the H+,K+-ATPase and SERCA, and the E2MgF structure of Na+,K+-ATPase with bound ouabain, but a continuous α-helical conformation in SERCA E2BeF21, and the latter fits well into the EM density map of H+,K+-ATPase (SCH)E2BeF (Supplementary Fig. S6). Thus, it is highly likely that this part of M2 continues as an α-helix beyond the cytoplasmic interface of the membrane. As the mutation of Q159 found at the cytoplasmic end of M2 has a fourfold lower affinity29 for SCH28080 despite its location on the opposite side of the membrane 35 Å away from the SCH28080-binding site (Fig. 4b,c), the conformational change of the A-M2 linker is important for the formation of the SCH28080-binding site. As suggested by SERCA structures21, the α-helix formation of the A-M2 linker may shorten the distance between the A domain and M2, inducing the observed location of the M1–M2 helix bundle (Fig. 3). Therefore, also for the H+,K+-ATPase, the A-M2 linker works as a determinant for the position of the M1–M2 bundle (Supplementary Fig. S6), which is likely to be coupled with the outwards movement of the M3–M4 helices.


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

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

SCH28080-induced conformational changes at the cytoplasmic part of the H+,K+-ATPase.(a–c) Close-up view of the A-M2 linker in E2AlF (a), (SCH)E2BeF (b) and their comparison (c). Each surface (white for E2AlF, blue for (SCH)E2BeF) and ribbon model (grey for E2AlF, colour for (SCH)E2BeF is as in Fig. 1a) represents the EM density map and homology model of the corresponding structures, respectively. The magenta sphere indicates Q159, which on mutation to Asn showed fourfold lower affinity for SCH28080. The A-M2 linker of E2AlF or (SCH)E2BeF is indicated by orange or blue arrows, respectively, yet the (SCH)E2BeF homology model does not contain this part (dotted blue line in b, c, see also text). Note that density for the N-terminal domain of the α-subunit (αNt) is absent in the (SCH)E2BeF structure (b, dotted circle) in contrast to the E2AlF structure (a). (d) Conformational change of the cytoplasmic domains. The surface shows the EM density map of (SCH)E2BeF with its homology model superimposed (colour codes as in Fig. 1a: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue). Orange mesh represents the E2AlF EM density map. It should be noted that the inter-subunit interaction between the P domain and the N-terminal tail of the β-subunit (βNt) observed in the E2AlF structure is not present in the (SCH)E2BeF structure (arrowhead, see text for details). All the EM density maps are drawn with a 1σ contour level. White arrows in all panels indicate observed conformational changes during the E2AlF→(SCH)E2BeF transition.
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f4: SCH28080-induced conformational changes at the cytoplasmic part of the H+,K+-ATPase.(a–c) Close-up view of the A-M2 linker in E2AlF (a), (SCH)E2BeF (b) and their comparison (c). Each surface (white for E2AlF, blue for (SCH)E2BeF) and ribbon model (grey for E2AlF, colour for (SCH)E2BeF is as in Fig. 1a) represents the EM density map and homology model of the corresponding structures, respectively. The magenta sphere indicates Q159, which on mutation to Asn showed fourfold lower affinity for SCH28080. The A-M2 linker of E2AlF or (SCH)E2BeF is indicated by orange or blue arrows, respectively, yet the (SCH)E2BeF homology model does not contain this part (dotted blue line in b, c, see also text). Note that density for the N-terminal domain of the α-subunit (αNt) is absent in the (SCH)E2BeF structure (b, dotted circle) in contrast to the E2AlF structure (a). (d) Conformational change of the cytoplasmic domains. The surface shows the EM density map of (SCH)E2BeF with its homology model superimposed (colour codes as in Fig. 1a: A domain, blue; P domain, green; N domain, yellow; TM domain, light blue). Orange mesh represents the E2AlF EM density map. It should be noted that the inter-subunit interaction between the P domain and the N-terminal tail of the β-subunit (βNt) observed in the E2AlF structure is not present in the (SCH)E2BeF structure (arrowhead, see text for details). All the EM density maps are drawn with a 1σ contour level. White arrows in all panels indicate observed conformational changes during the E2AlF→(SCH)E2BeF transition.
Mentions: The conformational change observed in the TM region is transmitted to the cytoplasmic part of the enzyme, mostly by connecting linker between the A domain and M2 (A-M2 linker, Fig. 4a–c). Because of the observed movement of the M1–M2 bundle on SCH28080 binding (Fig. 3b), the A-M2 linker forms an upright rod-like conformation separate from the P domain in the (SCH)E2BeF structure (Fig. 4b,c), which is in distinct contrast to its configuration in E2P* (Fig. 4a)1431. It should be noted that the A-M2 linker is missing in this (SCH)E2BeF homology model (Fig. 4b,c, dashed line, F161–P175), because of the large difference between the EM density map and the starting template structure (ouabain-bound Na+,K+-ATPase20). Comparison of the A-M2 linker conformations among several known structures of other P2-type ATPases202130, however, reveals that it can assume mainly two conformations (Supplementary Fig. S6). The A-M2 linker is unwound in structures of the E2AlF conformation of the H+,K+-ATPase and SERCA, and the E2MgF structure of Na+,K+-ATPase with bound ouabain, but a continuous α-helical conformation in SERCA E2BeF21, and the latter fits well into the EM density map of H+,K+-ATPase (SCH)E2BeF (Supplementary Fig. S6). Thus, it is highly likely that this part of M2 continues as an α-helix beyond the cytoplasmic interface of the membrane. As the mutation of Q159 found at the cytoplasmic end of M2 has a fourfold lower affinity29 for SCH28080 despite its location on the opposite side of the membrane 35 Å away from the SCH28080-binding site (Fig. 4b,c), the conformational change of the A-M2 linker is important for the formation of the SCH28080-binding site. As suggested by SERCA structures21, the α-helix formation of the A-M2 linker may shorten the distance between the A domain and M2, inducing the observed location of the M1–M2 helix bundle (Fig. 3). Therefore, also for the H+,K+-ATPase, the A-M2 linker works as a determinant for the position of the M1–M2 bundle (Supplementary Fig. S6), which is likely to be coupled with the outwards movement of the M3–M4 helices.

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