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Systematic comparison of molecular conformations of H+,K+-ATPase reveals an important contribution of the A-M2 linker for the luminal gating.

Abe K, Tani K, Fujiyoshi Y - J. Biol. Chem. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the Cellular and Structural Physiology Institute and Graduate School of Pharmaceutical Science, Nagoya University, Nagoya 464-8601, Japan kabe@cespi.nagoya-u.ac.jp.

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Conformational changes in the A-M2 linker.A–D, close-up view of the A-M2 linker (Glu-160–Gln-176, indicated by dark colors in homology models) in the reaction states of (SCH)E2·BeF (A), (SCH)E2·MgF (B), E2·AlF (C), and (Rb+)E2·AlF (D). Each molecular surface and ribbon model represents the EM density map (1σ) and homology model of the corresponding states, respectively. Black arrows show the position of the characteristic kink region found in the cytoplasmic portion of M1, which indicates the vertical shift of the M1M2 helices between the SCH28080-bound states (A and B) and SCH28080-free states (C and D). Dotted lines also serve as references for the vertical shift of the M1M2 helices. Although the EM density at the luminal portion of M1M2 is missing due to flexibility-induced disorder, the location of M1M2 is significantly different between the SCH28080-bound (A and B) and its free (C and D) conditions. The black arrowhead indicates the upright α-helical conformation of the A-M2 linker in the (SCH)E2·BeF state (A). The white arrowhead indicates the protruding EM density observed in the (SCH)E2·MgF state (B). The schematic representations of their conformational states are shown on the lower panel (see Fig. 9 for details).
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Figure 7: Conformational changes in the A-M2 linker.A–D, close-up view of the A-M2 linker (Glu-160–Gln-176, indicated by dark colors in homology models) in the reaction states of (SCH)E2·BeF (A), (SCH)E2·MgF (B), E2·AlF (C), and (Rb+)E2·AlF (D). Each molecular surface and ribbon model represents the EM density map (1σ) and homology model of the corresponding states, respectively. Black arrows show the position of the characteristic kink region found in the cytoplasmic portion of M1, which indicates the vertical shift of the M1M2 helices between the SCH28080-bound states (A and B) and SCH28080-free states (C and D). Dotted lines also serve as references for the vertical shift of the M1M2 helices. Although the EM density at the luminal portion of M1M2 is missing due to flexibility-induced disorder, the location of M1M2 is significantly different between the SCH28080-bound (A and B) and its free (C and D) conditions. The black arrowhead indicates the upright α-helical conformation of the A-M2 linker in the (SCH)E2·BeF state (A). The white arrowhead indicates the protruding EM density observed in the (SCH)E2·MgF state (B). The schematic representations of their conformational states are shown on the lower panel (see Fig. 9 for details).

Mentions: Rotation of the A domain was in turn transmitted to the TM region, which was mediated by the connecting linker between them (Fig. 7). The middle of the A-M2 linker, indicated as dark-colored tubes in Fig. 7, A–D, assumed an unwound loop structure in the (SCH)E2·MgF state (Fig. 7B) almost identical to that in the E2·AlF and (Rb+)E2·AlF states (Fig. 7, C and D, respectively), but significantly different from that in the (SCH)E2·BeF state (Fig. 7A). The conformation of the juxta-membranous portion of the A-M2 linker and M2 helix in (SCH)E2·MgF, however, was largely different from those in E2·AlF and (Rb+)E2·AlF, but is similar to that in (SCH)E2·BeF (Fig. 7A, Fig. 8). These differences were related to the conformational rearrangement of the A domain and TM helices (Figs. 3, 5). In particular, the luminal-open TM arrangement induced by SCH28080 binding was accompanied by a lateral shift of the M3M4 helices (Fig. 5) and at the same time a vertical shift of M1M2 helices toward the luminal side, as seen in the different vertical orientations of the kink region of the A-M1 linkers (Fig. 7, A–D, black arrows) and the characteristic protrusion of the EM density at the end of M2 in the (SCH)E2·MgF state (Figs. 7B and 8B, white arrowhead). In the (SCH)E2·MgF state, the cytoplasmic portion assumed a typical “luminal-closed” form (∼20° rotated A domain and unwound loop structure of the A-M2 linker), which might subsequently induce the luminal gate closure in the absence of SCH28080 as seen in the E2·AlF structure (Fig. 5C). The M1M2 helices, however, could not move due to the luminal-open conformation of the TM helices fixed by bound SCH28080 at the luminal cavity. As a result, the (SCH)E2·MgF structure represents a hybrid conformation, with the relative orientation of the cytoplasmic domains similar to that in a typical luminal-closed type conformation (Figs. 3 and 7) and the TM region assuming a luminal-open conformation (Fig. 5).


Systematic comparison of molecular conformations of H+,K+-ATPase reveals an important contribution of the A-M2 linker for the luminal gating.

Abe K, Tani K, Fujiyoshi Y - J. Biol. Chem. (2014)

Conformational changes in the A-M2 linker.A–D, close-up view of the A-M2 linker (Glu-160–Gln-176, indicated by dark colors in homology models) in the reaction states of (SCH)E2·BeF (A), (SCH)E2·MgF (B), E2·AlF (C), and (Rb+)E2·AlF (D). Each molecular surface and ribbon model represents the EM density map (1σ) and homology model of the corresponding states, respectively. Black arrows show the position of the characteristic kink region found in the cytoplasmic portion of M1, which indicates the vertical shift of the M1M2 helices between the SCH28080-bound states (A and B) and SCH28080-free states (C and D). Dotted lines also serve as references for the vertical shift of the M1M2 helices. Although the EM density at the luminal portion of M1M2 is missing due to flexibility-induced disorder, the location of M1M2 is significantly different between the SCH28080-bound (A and B) and its free (C and D) conditions. The black arrowhead indicates the upright α-helical conformation of the A-M2 linker in the (SCH)E2·BeF state (A). The white arrowhead indicates the protruding EM density observed in the (SCH)E2·MgF state (B). The schematic representations of their conformational states are shown on the lower panel (see Fig. 9 for details).
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Figure 7: Conformational changes in the A-M2 linker.A–D, close-up view of the A-M2 linker (Glu-160–Gln-176, indicated by dark colors in homology models) in the reaction states of (SCH)E2·BeF (A), (SCH)E2·MgF (B), E2·AlF (C), and (Rb+)E2·AlF (D). Each molecular surface and ribbon model represents the EM density map (1σ) and homology model of the corresponding states, respectively. Black arrows show the position of the characteristic kink region found in the cytoplasmic portion of M1, which indicates the vertical shift of the M1M2 helices between the SCH28080-bound states (A and B) and SCH28080-free states (C and D). Dotted lines also serve as references for the vertical shift of the M1M2 helices. Although the EM density at the luminal portion of M1M2 is missing due to flexibility-induced disorder, the location of M1M2 is significantly different between the SCH28080-bound (A and B) and its free (C and D) conditions. The black arrowhead indicates the upright α-helical conformation of the A-M2 linker in the (SCH)E2·BeF state (A). The white arrowhead indicates the protruding EM density observed in the (SCH)E2·MgF state (B). The schematic representations of their conformational states are shown on the lower panel (see Fig. 9 for details).
Mentions: Rotation of the A domain was in turn transmitted to the TM region, which was mediated by the connecting linker between them (Fig. 7). The middle of the A-M2 linker, indicated as dark-colored tubes in Fig. 7, A–D, assumed an unwound loop structure in the (SCH)E2·MgF state (Fig. 7B) almost identical to that in the E2·AlF and (Rb+)E2·AlF states (Fig. 7, C and D, respectively), but significantly different from that in the (SCH)E2·BeF state (Fig. 7A). The conformation of the juxta-membranous portion of the A-M2 linker and M2 helix in (SCH)E2·MgF, however, was largely different from those in E2·AlF and (Rb+)E2·AlF, but is similar to that in (SCH)E2·BeF (Fig. 7A, Fig. 8). These differences were related to the conformational rearrangement of the A domain and TM helices (Figs. 3, 5). In particular, the luminal-open TM arrangement induced by SCH28080 binding was accompanied by a lateral shift of the M3M4 helices (Fig. 5) and at the same time a vertical shift of M1M2 helices toward the luminal side, as seen in the different vertical orientations of the kink region of the A-M1 linkers (Fig. 7, A–D, black arrows) and the characteristic protrusion of the EM density at the end of M2 in the (SCH)E2·MgF state (Figs. 7B and 8B, white arrowhead). In the (SCH)E2·MgF state, the cytoplasmic portion assumed a typical “luminal-closed” form (∼20° rotated A domain and unwound loop structure of the A-M2 linker), which might subsequently induce the luminal gate closure in the absence of SCH28080 as seen in the E2·AlF structure (Fig. 5C). The M1M2 helices, however, could not move due to the luminal-open conformation of the TM helices fixed by bound SCH28080 at the luminal cavity. As a result, the (SCH)E2·MgF structure represents a hybrid conformation, with the relative orientation of the cytoplasmic domains similar to that in a typical luminal-closed type conformation (Figs. 3 and 7) and the TM region assuming a luminal-open conformation (Fig. 5).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: From the Cellular and Structural Physiology Institute and Graduate School of Pharmaceutical Science, Nagoya University, Nagoya 464-8601, Japan kabe@cespi.nagoya-u.ac.jp.

Show MeSH