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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 change of H+,K+-ATPase monitored by FITC fluorescence.A, dose-dependent inhibition of K+-p-nitrophenyl phosphatase activity of FITC-modified H+,K+-ATPase by XFs. Membrane-bound FITC-modified H+,K+-ATPase preparations were incubated with the indicated concentrations of BeF (red), AlF (green), or MgF (yellow), and their K+-p-nitrophenyl phosphatase activity was measured (filled circles and lines). Open symbols with dotted lines indicate the dose dependence of XFs on H+,K+-ATPase activity of the unmodified enzyme (17). The lower table indicates apparent the IC50 for each XF used. The values are the mean ± S.D. (n = 3) when larger than the symbol. B and C, EM maps of the cytoplasmic domains of H+,K+-ATPase in the (SCH)E2·BeF (B) and that in (SCH)E2·MgF states (C), superimposed with their respective homology models (color coded as in Fig. 2). EM density responsible for bound ADP (stick) is clearly seen in the (SCH)E2·BeF state but missing in the (SCH)E2·MgF state (black arrowheads). The FITC-binding site (Lys-518) at the nucleotide binding pocket is indicated by spheres in each map. Schematic representations of each conformational state are shown on the upper left (see Fig. 9 for details). D and E, changes in FITC fluorescence intensities in response to the addition of indicated ligands. D, a representative of the time-course experiment of FITC fluorescence intensity. FITC-modified H+,K+-ATPase membrane fractions were incubated at 37 °C, and their fluorescence intensity changes (ΔF, scale for 20% change is shown in the right) were monitored. White and black arrowheads indicate time of the addition of phosphate, its analogs, or SCH28080 (SCH) to the sample, respectively. E, amount of ΔF after the addition of the indicated reagents is shown. Open and closed columns indicate the absence or presence of SCH28080, respectively, with the indicated phosphate (analogs) added as shown at the bottom. Data displayed show the mean ± S.D. from three independent experiments. Expected reaction states are shown in parentheses.
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Figure 4: Conformational change of H+,K+-ATPase monitored by FITC fluorescence.A, dose-dependent inhibition of K+-p-nitrophenyl phosphatase activity of FITC-modified H+,K+-ATPase by XFs. Membrane-bound FITC-modified H+,K+-ATPase preparations were incubated with the indicated concentrations of BeF (red), AlF (green), or MgF (yellow), and their K+-p-nitrophenyl phosphatase activity was measured (filled circles and lines). Open symbols with dotted lines indicate the dose dependence of XFs on H+,K+-ATPase activity of the unmodified enzyme (17). The lower table indicates apparent the IC50 for each XF used. The values are the mean ± S.D. (n = 3) when larger than the symbol. B and C, EM maps of the cytoplasmic domains of H+,K+-ATPase in the (SCH)E2·BeF (B) and that in (SCH)E2·MgF states (C), superimposed with their respective homology models (color coded as in Fig. 2). EM density responsible for bound ADP (stick) is clearly seen in the (SCH)E2·BeF state but missing in the (SCH)E2·MgF state (black arrowheads). The FITC-binding site (Lys-518) at the nucleotide binding pocket is indicated by spheres in each map. Schematic representations of each conformational state are shown on the upper left (see Fig. 9 for details). D and E, changes in FITC fluorescence intensities in response to the addition of indicated ligands. D, a representative of the time-course experiment of FITC fluorescence intensity. FITC-modified H+,K+-ATPase membrane fractions were incubated at 37 °C, and their fluorescence intensity changes (ΔF, scale for 20% change is shown in the right) were monitored. White and black arrowheads indicate time of the addition of phosphate, its analogs, or SCH28080 (SCH) to the sample, respectively. E, amount of ΔF after the addition of the indicated reagents is shown. Open and closed columns indicate the absence or presence of SCH28080, respectively, with the indicated phosphate (analogs) added as shown at the bottom. Data displayed show the mean ± S.D. from three independent experiments. Expected reaction states are shown in parentheses.

Mentions: The fluorescence probe FITC preferentially forms a covalent bond with the ϵ-amino group of the Lys-518 residue, which is embedded in the conserved Lys-518 in the ATP binding site of the N domain (38). This chemical modification of the Lys residue impairs H+,K+-ATPase activity (1.7% of residual H+,K+-ATPase activity compared with that of mock-treated enzyme) due to a loss of ATP-binding ability, suggesting that the FITC probe is located at the nucleotide binding position. The FITC-modified H+,K+-ATPase, however, can hydrolyze substrates less bulky than ATP, such as acetyl phosphate or p-nitrophenyl phosphate (39), showing 76% residual K+-p-nitrophenyl phosphatase activity compared with that of mock-treated enzyme. The FITC-modified enzyme also has affinities for XFs comparable with those of the unmodified enzyme (Fig. 4A). Therefore, the FITC-modified enzyme remains active and undergoes a conformational change in response to substrate binding, allowing us to monitor the conformational changes, especially those occurring at the nucleotide binding site in the N domain, based on the fluorescence intensity (40).


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 change of H+,K+-ATPase monitored by FITC fluorescence.A, dose-dependent inhibition of K+-p-nitrophenyl phosphatase activity of FITC-modified H+,K+-ATPase by XFs. Membrane-bound FITC-modified H+,K+-ATPase preparations were incubated with the indicated concentrations of BeF (red), AlF (green), or MgF (yellow), and their K+-p-nitrophenyl phosphatase activity was measured (filled circles and lines). Open symbols with dotted lines indicate the dose dependence of XFs on H+,K+-ATPase activity of the unmodified enzyme (17). The lower table indicates apparent the IC50 for each XF used. The values are the mean ± S.D. (n = 3) when larger than the symbol. B and C, EM maps of the cytoplasmic domains of H+,K+-ATPase in the (SCH)E2·BeF (B) and that in (SCH)E2·MgF states (C), superimposed with their respective homology models (color coded as in Fig. 2). EM density responsible for bound ADP (stick) is clearly seen in the (SCH)E2·BeF state but missing in the (SCH)E2·MgF state (black arrowheads). The FITC-binding site (Lys-518) at the nucleotide binding pocket is indicated by spheres in each map. Schematic representations of each conformational state are shown on the upper left (see Fig. 9 for details). D and E, changes in FITC fluorescence intensities in response to the addition of indicated ligands. D, a representative of the time-course experiment of FITC fluorescence intensity. FITC-modified H+,K+-ATPase membrane fractions were incubated at 37 °C, and their fluorescence intensity changes (ΔF, scale for 20% change is shown in the right) were monitored. White and black arrowheads indicate time of the addition of phosphate, its analogs, or SCH28080 (SCH) to the sample, respectively. E, amount of ΔF after the addition of the indicated reagents is shown. Open and closed columns indicate the absence or presence of SCH28080, respectively, with the indicated phosphate (analogs) added as shown at the bottom. Data displayed show the mean ± S.D. from three independent experiments. Expected reaction states are shown in parentheses.
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Figure 4: Conformational change of H+,K+-ATPase monitored by FITC fluorescence.A, dose-dependent inhibition of K+-p-nitrophenyl phosphatase activity of FITC-modified H+,K+-ATPase by XFs. Membrane-bound FITC-modified H+,K+-ATPase preparations were incubated with the indicated concentrations of BeF (red), AlF (green), or MgF (yellow), and their K+-p-nitrophenyl phosphatase activity was measured (filled circles and lines). Open symbols with dotted lines indicate the dose dependence of XFs on H+,K+-ATPase activity of the unmodified enzyme (17). The lower table indicates apparent the IC50 for each XF used. The values are the mean ± S.D. (n = 3) when larger than the symbol. B and C, EM maps of the cytoplasmic domains of H+,K+-ATPase in the (SCH)E2·BeF (B) and that in (SCH)E2·MgF states (C), superimposed with their respective homology models (color coded as in Fig. 2). EM density responsible for bound ADP (stick) is clearly seen in the (SCH)E2·BeF state but missing in the (SCH)E2·MgF state (black arrowheads). The FITC-binding site (Lys-518) at the nucleotide binding pocket is indicated by spheres in each map. Schematic representations of each conformational state are shown on the upper left (see Fig. 9 for details). D and E, changes in FITC fluorescence intensities in response to the addition of indicated ligands. D, a representative of the time-course experiment of FITC fluorescence intensity. FITC-modified H+,K+-ATPase membrane fractions were incubated at 37 °C, and their fluorescence intensity changes (ΔF, scale for 20% change is shown in the right) were monitored. White and black arrowheads indicate time of the addition of phosphate, its analogs, or SCH28080 (SCH) to the sample, respectively. E, amount of ΔF after the addition of the indicated reagents is shown. Open and closed columns indicate the absence or presence of SCH28080, respectively, with the indicated phosphate (analogs) added as shown at the bottom. Data displayed show the mean ± S.D. from three independent experiments. Expected reaction states are shown in parentheses.
Mentions: The fluorescence probe FITC preferentially forms a covalent bond with the ϵ-amino group of the Lys-518 residue, which is embedded in the conserved Lys-518 in the ATP binding site of the N domain (38). This chemical modification of the Lys residue impairs H+,K+-ATPase activity (1.7% of residual H+,K+-ATPase activity compared with that of mock-treated enzyme) due to a loss of ATP-binding ability, suggesting that the FITC probe is located at the nucleotide binding position. The FITC-modified H+,K+-ATPase, however, can hydrolyze substrates less bulky than ATP, such as acetyl phosphate or p-nitrophenyl phosphate (39), showing 76% residual K+-p-nitrophenyl phosphatase activity compared with that of mock-treated enzyme. The FITC-modified enzyme also has affinities for XFs comparable with those of the unmodified enzyme (Fig. 4A). Therefore, the FITC-modified enzyme remains active and undergoes a conformational change in response to substrate binding, allowing us to monitor the conformational changes, especially those occurring at the nucleotide binding site in the N domain, based on the fluorescence intensity (40).

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