Conformational changes in gastric H+/K+-ATPase monitored by difference Fourier-transform infrared spectroscopy and hydrogen/deuterium exchange.
Gastric H+/K+-ATPase is a P-type ATPase responsible for acid secretion in the stomach. This protein adopts mainly two conformations called E1 and E2. Even though two high-resolution structures for a P-ATPase in these conformations are available, little structural information is available about the transition between these two conformations. In the present study, we used two experimental approaches to investigate the structural differences that occur when gastric ATPase is placed in the presence of various ligands and ligand combinations. We used attenuated total reflection-Fourier-transform IR experiments under a flowing buffer to modify the environment of the protein inside the measurement cell. The high accuracy of the results allowed us to demonstrate that the E1-E2 transition induces a net change in the secondary structure that concerns 10-15 amino acid residues of a total of 1324 in the proteins. The E2.K+ structure is characterized by a decreased beta-sheet content and an increase in the disordered structure content with respect to the E1 form of the enzyme. Modifications in the absorption of the side chain of amino acids are also suggested. By using hydrogen/deuterium-exchange kinetics, we show that tertiary-structure modifications occurred in the presence of the same ligands, but these changes involved several hundreds of residues. The present study suggests that conformational changes in the catalytic cycle imply secondary-structure rearrangements of small hinge regions that have an impact on large domain re-organizations.
Scheirlinckx
Frantz
F
Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Free University of Brussels, CP206/2, Boulevard du Triomphe, B-1050 Brussels, Belgium.
Raussens
Vincent
V
Ruysschaert
Jean-Marie
JM
Goormaghtigh
Erik
E
D000818:Animals; D041961:Deuterium Exchange Measurement / Q000379:methods*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013270:Stomach / Q000201:enzymology*; D013552:Swine
D017506:H(+)-K(+)-Exchanging ATPase