Systematic comparison of molecular conformations of H+,K+-ATPase reveals an important contribution of the A-M2 linker for the luminal gating.
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.
Affiliation: From the Cellular and Structural Physiology Institute and Graduate School of Pharmaceutical Science, Nagoya University, Nagoya 464-8601, Japan firstname.lastname@example.org.Show MeSH
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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).
Affiliation: From the Cellular and Structural Physiology Institute and Graduate School of Pharmaceutical Science, Nagoya University, Nagoya 464-8601, Japan email@example.com.