Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the α-chain of pig gastric H+/K+-ATPase

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Abstract

It has been well established that phosphorylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the α-chain of pig gastric H+/K+-ATPase, reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H+/K+-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation, and the peptide adopted two α-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H+/K+-ATPase.

Section snippets

Materials and methods

Peptide preparation. Non-phospho- and phospho-peptides containing the 33 N-terminal amino acids of pig gastric H+/K+-ATPase (Gly2–Gly34) based on the sequence of the pig H+/K+-ATPase α-chain, i.e., N33np (GKAENYELYQVELGPGPSGDMAAKMSKKKAGRG; 2–34), and N33pY10 that had been phosphorylated at Tyr 10 of N33np, were synthesized by Sawady Technology (Tokyo, Japan).

Circular dichroism. All measurements were performed on a Jasco J-725 spectropolarimeter (Jasco, Japan). Spectra were recorded at 25 °C and

Structure determination of the N-terminal fragment of the α-chain of pig gastric H+/K+-ATPase

In order to reveal the conformation of the N-terminal region of the α-chain of pig gastric H+/K+-ATPase, two peptides, N33np and N33pY10, were synthesized. The former contained the N-terminal region of pig gastric H+/K+-ATPase (Gly 2–Gly 34) without the phosphate group at Tyr 10, and the latter contained the same region but with the phosphate group at Tyr 10. Because the phosphorylation of Tyr 10 occurs before the phosphorylation of Tyr 7 via an endogenous Tyr-kinase [6], we designed a

Acknowledgements

We thank Professor Shin-ichi Tate of Japan Advanced Institute of Science and Technology for offering NMR experimental time and their useful discussions. We also thank Dr. Yoshihiro Mori of Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University for supporting CD experiments. This work was supported by Grants-in-Aid for Scientific Research (10308028) and the International Scientific Research Program (10044048) from the Ministry of Education, Science and Culture of Japan,

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