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 email@example.com.Show MeSH
Mentions: Previously, we reported several cryo-EM structures of H+,K+-ATPase bound to different XFs in the presence or absence of ions and substrates for the TM domain (such as transported cation K+, its congener Rb+, and a specific antagonist SCH28080), which include the reaction state analog of E2·BeF (17), E2·AlF (13), (SCH)E2·BeF (18), and (Rb+)E2·AlF (19). For systematic comparison of the molecular conformations induced by XFs mimicking the reaction substeps of E2P dephosphorylation, we obtained two-dimensional crystals in the presence of SCH28080 in combination with either AlF or MgF and determined their three-dimensional structures at 8 Å and 7 Å resolution, respectively (Fig. 2A, Table 1). It is notable that no two-dimensional crystals were produced in the presence of either MgF alone or MgF with Rb+ (not shown). Because inhibition of H+,K+-ATPase activity by MgF is reversible and weaker than that of other XFs (17), stabilization of the molecular conformation by SCH28080 is required for the two-dimensional crystal formation when MgF is used as the phosphate analog. The two newly determined structures, however, were almost identical in their overall conformation, including the arrangement of the TM helices, the azimuthal position of the A domain, and the connecting linker between them (Fig. 2, A and B). In fact, x-ray structures of SERCA in the E2·AlF and E2·MgF states also indicated identical molecular conformations (21). Therefore, we concluded that these two structures of H+,K+-ATPase adopt an indistinguishable conformation and thus used the (SCH)E2·MgF structure as a representative with better crystallographic characteristics for subsequent comparison of the molecular conformation with other cryo-EM structures.
Affiliation: From the Cellular and Structural Physiology Institute and Graduate School of Pharmaceutical Science, Nagoya University, Nagoya 464-8601, Japan firstname.lastname@example.org.