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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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SCH28080 binding to the H+,K+-ATPase.A and B, close-up view of the SCH28080-binding site at the luminal cavity in the (SCH)E2·BeF (A) and (SCH)E2·MgF (B) states. Molecular surface representation of each EM density map (1σ) with superimposed homology models (color gradually changes from M1 (blue) to M10 (red) as indicated). Cross-sections of the SCH28080-binding site are shown as a light blue surface. The figures were viewed from parallel to the membrane plane (cytoplasmic side-up). C, dose dependence of SCH28080 binding to the E2P and its analog states. Purified H+,K+-ATPase membrane fractions were incubated in the presence of the indicated ligands followed by the addition of [3H]SCH28080. Data shown are the mean ± S.E. (n = 3), measured at 25 °C. The inset table shows the affinity (Kd) ± S.E. for the SCH28080 binding in each indicated condition. D, van't Hoff plot for SCH28080 binding. Kd values were determined directly from three independent binding experiments at each temperature (K). Colors for each symbol are as in C. Values are the mean ± S.E. (n = 3). E, the contribution of enthalpy and entropy to the free energy of SCH28080 binding. Values for enthalpy (ΔH, red columns) and entropy (ΔS, green columns) were obtained from the van't Hoff relationship (Equation 1) for each indicated condition, as described under “Experimental Procedures.” These values were then used to calculate the corresponding free energies (ΔG, blue columns, calculated as 25 °C) using Equation 2.
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Figure 6: SCH28080 binding to the H+,K+-ATPase.A and B, close-up view of the SCH28080-binding site at the luminal cavity in the (SCH)E2·BeF (A) and (SCH)E2·MgF (B) states. Molecular surface representation of each EM density map (1σ) with superimposed homology models (color gradually changes from M1 (blue) to M10 (red) as indicated). Cross-sections of the SCH28080-binding site are shown as a light blue surface. The figures were viewed from parallel to the membrane plane (cytoplasmic side-up). C, dose dependence of SCH28080 binding to the E2P and its analog states. Purified H+,K+-ATPase membrane fractions were incubated in the presence of the indicated ligands followed by the addition of [3H]SCH28080. Data shown are the mean ± S.E. (n = 3), measured at 25 °C. The inset table shows the affinity (Kd) ± S.E. for the SCH28080 binding in each indicated condition. D, van't Hoff plot for SCH28080 binding. Kd values were determined directly from three independent binding experiments at each temperature (K). Colors for each symbol are as in C. Values are the mean ± S.E. (n = 3). E, the contribution of enthalpy and entropy to the free energy of SCH28080 binding. Values for enthalpy (ΔH, red columns) and entropy (ΔS, green columns) were obtained from the van't Hoff relationship (Equation 1) for each indicated condition, as described under “Experimental Procedures.” These values were then used to calculate the corresponding free energies (ΔG, blue columns, calculated as 25 °C) using Equation 2.

Mentions: Despite the identical luminal-open arrangement of TM helices in the (SCH)E2·BeF and (SCH)E2·MgF states, however, most of the SCH28080 moiety was invisible in the density map of (SCH)E2·MgF (Fig. 6B), which is in marked contrast to that of (SCH)E2·BeF (Fig. 6A). Because SCH28080 was required for the two-dimensional crystallization in the (SCH)E2·MgF condition as well as the different arrangement of the TM helices in (SCH)E2·AlF from its absent E2·AlF state (Fig. 5C), the observed poor density at the SCH28080-binding site was likely due to the disorder of this antagonist at the luminal cavity. Such a difference around the presumed SCH28080 density in the (SCH)E2·BeF and the (SCH)E2·MgF states prompted us to investigate the SCH28080 binding affinity to H+,K+-ATPase bound to the various XFs (Fig. 6C). Using 3H-labeled SCH28080, the effects of XFs on the SCH28080 binding affinity were examined (41). BeF-bound H+,K+-ATPase had a high affinity for the SCH28080 binding (Kd = 14.8 ± 1.1 nm), which was as high as that for the genuine E2P formed from MgPi (Kd = 13.4 ± 1.4 nm) at 25 °C. In contrast, AlF- or MgF-bound H+,K+-ATPase had 3∼4 times lower affinity for SCH28080 (Kd = 33.6 ± 2.2 nm, Kd = 54 ± 4.5 nm) than BeF-bound H+,K+-ATPase, consistent with the observed poor density of SCH28080 at the luminal cavity in the (SCH)E2·MgF (Fig. 6B) and (SCH)E2·AlF (not shown) states.


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)

SCH28080 binding to the H+,K+-ATPase.A and B, close-up view of the SCH28080-binding site at the luminal cavity in the (SCH)E2·BeF (A) and (SCH)E2·MgF (B) states. Molecular surface representation of each EM density map (1σ) with superimposed homology models (color gradually changes from M1 (blue) to M10 (red) as indicated). Cross-sections of the SCH28080-binding site are shown as a light blue surface. The figures were viewed from parallel to the membrane plane (cytoplasmic side-up). C, dose dependence of SCH28080 binding to the E2P and its analog states. Purified H+,K+-ATPase membrane fractions were incubated in the presence of the indicated ligands followed by the addition of [3H]SCH28080. Data shown are the mean ± S.E. (n = 3), measured at 25 °C. The inset table shows the affinity (Kd) ± S.E. for the SCH28080 binding in each indicated condition. D, van't Hoff plot for SCH28080 binding. Kd values were determined directly from three independent binding experiments at each temperature (K). Colors for each symbol are as in C. Values are the mean ± S.E. (n = 3). E, the contribution of enthalpy and entropy to the free energy of SCH28080 binding. Values for enthalpy (ΔH, red columns) and entropy (ΔS, green columns) were obtained from the van't Hoff relationship (Equation 1) for each indicated condition, as described under “Experimental Procedures.” These values were then used to calculate the corresponding free energies (ΔG, blue columns, calculated as 25 °C) using Equation 2.
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Figure 6: SCH28080 binding to the H+,K+-ATPase.A and B, close-up view of the SCH28080-binding site at the luminal cavity in the (SCH)E2·BeF (A) and (SCH)E2·MgF (B) states. Molecular surface representation of each EM density map (1σ) with superimposed homology models (color gradually changes from M1 (blue) to M10 (red) as indicated). Cross-sections of the SCH28080-binding site are shown as a light blue surface. The figures were viewed from parallel to the membrane plane (cytoplasmic side-up). C, dose dependence of SCH28080 binding to the E2P and its analog states. Purified H+,K+-ATPase membrane fractions were incubated in the presence of the indicated ligands followed by the addition of [3H]SCH28080. Data shown are the mean ± S.E. (n = 3), measured at 25 °C. The inset table shows the affinity (Kd) ± S.E. for the SCH28080 binding in each indicated condition. D, van't Hoff plot for SCH28080 binding. Kd values were determined directly from three independent binding experiments at each temperature (K). Colors for each symbol are as in C. Values are the mean ± S.E. (n = 3). E, the contribution of enthalpy and entropy to the free energy of SCH28080 binding. Values for enthalpy (ΔH, red columns) and entropy (ΔS, green columns) were obtained from the van't Hoff relationship (Equation 1) for each indicated condition, as described under “Experimental Procedures.” These values were then used to calculate the corresponding free energies (ΔG, blue columns, calculated as 25 °C) using Equation 2.
Mentions: Despite the identical luminal-open arrangement of TM helices in the (SCH)E2·BeF and (SCH)E2·MgF states, however, most of the SCH28080 moiety was invisible in the density map of (SCH)E2·MgF (Fig. 6B), which is in marked contrast to that of (SCH)E2·BeF (Fig. 6A). Because SCH28080 was required for the two-dimensional crystallization in the (SCH)E2·MgF condition as well as the different arrangement of the TM helices in (SCH)E2·AlF from its absent E2·AlF state (Fig. 5C), the observed poor density at the SCH28080-binding site was likely due to the disorder of this antagonist at the luminal cavity. Such a difference around the presumed SCH28080 density in the (SCH)E2·BeF and the (SCH)E2·MgF states prompted us to investigate the SCH28080 binding affinity to H+,K+-ATPase bound to the various XFs (Fig. 6C). Using 3H-labeled SCH28080, the effects of XFs on the SCH28080 binding affinity were examined (41). BeF-bound H+,K+-ATPase had a high affinity for the SCH28080 binding (Kd = 14.8 ± 1.1 nm), which was as high as that for the genuine E2P formed from MgPi (Kd = 13.4 ± 1.4 nm) at 25 °C. In contrast, AlF- or MgF-bound H+,K+-ATPase had 3∼4 times lower affinity for SCH28080 (Kd = 33.6 ± 2.2 nm, Kd = 54 ± 4.5 nm) than BeF-bound H+,K+-ATPase, consistent with the observed poor density of SCH28080 at the luminal cavity in the (SCH)E2·MgF (Fig. 6B) and (SCH)E2·AlF (not shown) states.

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
Related in: MedlinePlus