Structural and functional characterization of H+, K+-ATPase with bound fluorinated phosphate analogs.
Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. In order to characterize the phosphorylation events on H(+),K(+)-ATPase, the properties of fluorinated phosphate analogs [XFs, e.g. aluminum fluoride (AlF), beryllium fluoride (BeF) and magnesium fluoride (MgF)], and the structural differences induced by XFs were investigated. The addition of divalent cations to the XF-inhibited H(+),K(+)-ATPase restores the activity of the AlF- or MgF-inhibited, but not of the BeF-inhibited enzyme, although limited trypsin digestion reveals that they assume the same E(2)P-like state. To clarify the conformational differences induced by XFs, the structure of BeF-bound H(+),K(+)-ATPase was analyzed at 8A resolution. The structure is almost identical to the previously reported AlF-bound E(2)P structure, unlike the distinctive X-ray structure of BeF-bound SERCA, in which the luminal gate was observed to be widely opened. Since the analyzed structure of the H(+),K(+)-ATPase revealed that both AlF and BeF-bound to the P domain were not exposed to the solvent, the dissociation of XFs induced by divalent cations could be interpreted in terms of stability against thermal fluctuations. Furthermore, the conformational differences found between the cytoplasmic domains of H(+),K(+)-ATPase and SERCA provide a framework to understand the characteristic mechanism, by which divalent cations reactivate the XF-inhibited H(+),K(+)-ATPase.
Abe
Kazuhiro
K
Department of Biophysics, Faculty of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-0852, Japan. ikkei@em.biophys.kyoto-u.ac.jp
Tani
Kazutoshi
K
Fujiyoshi
Yoshinori
Y
D020285:Cryoelectron Microscopy; D003460:Crystallization; D005459:Fluorides / Q000737:chemistry; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry*; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation
D010710:Phosphates; D017506:H(+)-K(+)-Exchanging ATPase; D005459:Fluorides