Conformational changes in gastric H+/K+-ATPase monitored by difference Fourier-transform infrared spectroscopy and hydrogen/deuterium exchange (original) (raw)
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European Journal of Biochemistry, 2001
Abbreviations: ATR, attenuated total reflection; FITC, fluoresceine isothiocyanate; FTIR, fourier transform infrared spectroscopy; PCM, 7-diethylamino-3-(4 0 -maleimidylphenyl)-4-methylcoumarine; MDPQ, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo(3,2-c)quinoline; Mdazip, 8-[(4-azidophenyl)methoxy]-1-trithiomethyl-2,3-dimethylimidazo-(1,2-a)pyrimidium iodide. Eur. J. Biochem. 268, 5135-5141 (2001) q FEBS 2001 q FEBS 2001 Structural modification of the H 1 /K 1 -ATPase (Eur. J. Biochem. 268) 5139 q FEBS 2001 Structural modification of the H 1 /K 1 -ATPase (Eur. J. Biochem. 268) 5141
Structural Aspects of the Gastric H, K ATPase
Annals of The New York Academy of Sciences, 1997
The gastric H,K-ATPase is an alpha,beta heterodimer. The large catalytic subunit is composed, in the case of the hog enzyme, of 1033 amino acids, whereas the beta subunit is composed of about 291 amino acids and is heavily glycosylated. The membrane topology of the alpha subunit is difficult to predict using hydropathy analysis. Tryptic hydrolysis of intact, inside out vesicles followed by cysteine labelling with fluorescein-5-maleimide provided experimental evidence for an 8 membrane spanning model for the alpha subunit, between residues 104 and 162 (M1/M2), 291 and 358 (M3/M4), 776 and 835 (M5/M6), and 853 and 946 (M7/MS). No evidence was found for a pair of segments (M9/M10) towards the C terminal end of the molecule, contrary to predictions for the Na,K-and Ca-ATPases. Iodination of intact vesicles followed by carboxypeptidase Y cleavage of the C terminal tyrosines showed that the C terminal end of the alpha subunit was cytoplasmic. The epitope for antibody 146 was extracytoplasmic and located between residues 871 to 874 between M7/M8. The binding site of the K competitive imidazo-pyridine, SCH28080, was to the extracytoplasmic loop between M 1 and M2, whereas the binding of the covalent SH reagent generated from acid activation of omeprazole in acid transporting vesicles was to 2 cysteines at positions 813 (or 822) and 892 predicted to be in the extracytoplasmic loops connecting M5/M6 and M7/M8, respectively. The beta subunit was only hydrolysed in broken vesicles. A fragment beginning at position 236 was liberated under these conditions only in the presence of reducing agents, showing that cysteine 210 and 263 were disulfide linked. It seems that this subunit has only a single membrane spanning segment as predicted by hydrophobicity. Binding of either SCH28080 or omeprazole to the extracytoplasmic face of the enzyme affected cytoplasmic conformational changes, showing that there was transmembranal transmission of changes of shape of the protein.
Structural interactions between a- and β-subunits of the gastric H,K-ATPase
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
Structural and functional interactions between a-and /3-subunits of the H,K-ATPase were explored. The sensitivity to trypsinolysis of c~-subunit was monitored by SDS-PAGE in control H,K-ATPase-enriched microsomes and in microsomes in which disulfide bonds of the /3-subunit were reduced using 2-mercaptoethanol (2-ME). Reduction of /3-subunit disulfide bonds increased the susceptibility of the c~-subunit to tryptic digestion. Kinetics of trypsinolysis were also carried out in the presence of ligands known to bind with H,K-ATPase and favor a particular conformer state in the native enzyme. The time-course for release of tryptic peptides was monitored in protein stained gels and Western blots probed with monoclonal antibody a-H,K,12.18. In control preparations, where /3-subunit disulfides remained intact, trypsinolysis in the presence of ATP or K ÷ produced distinctive patterns of tryptic fragments, each characteristic of the conformational states induced by the respective ligand. For 2-ME-treated microsomes the altered a-subunit was unable to undergo ligand-induced conformational changes. The increased susceptibility of the a-subunit to trypsinization, the change in accessibility of tryptic cleavage sites and the inability of the a-subunit to undergo ligand-induced conformational changes after reduction of the/3-subunit disulfides suggest that the interactions between a-and /3-subunits are important for the conformational stability of the functional holoenzyme. A model localizing the most susceptible tryptic cleavage sites in control and 2-ME-reduced states is presented.
Analysis of the membrane domain of the gastric H+/K+-ATPase
Journal of Experimental Biology
An ion pump gets its name from its ability to move ions uphill, or against a concentration gradient, and requiring input of energy across a membrane that is an asymmetric phospholipid bilayer. The mechanical pumps created by man, such as those powered by rotary pistons, linear motors or other devices, may reflect the biological choice for such devices. A pump, biological or mechanical, must derive energy from some source. A biological pump derives energy from the breakdown of substrates, such as by oxidation or hydrolysis, from absorption of light or from ATP. These scalar processes are converted to the vectorial property of transport by the orientation of the protein across a membrane bilayer, whence conformational changes induced by energization in the protein are translated into transmembrane ion motion either with rotation, as in the F1Fo or V-ATPases, or by membrane helix tilt, as in rhodopsins or P-ATPases.
European journal of biochemistry / FEBS, 1998
Models of P-type ATPase predict that membrane-embedded fragments represent about 20% of the protein and adopt an all-alpha-helical structure. While this prediction was confirmed for the Ca2-ATPase [Corbalan-Garcia, S., Teruel, J., Villalain, J. & Gomez-Fernandez, J. (1994) Biochemistry 33, 8247-8254], it is at odds with recent experimental evidence gathered on the Neurospora crassa plasma membrane H+-ATPase [Vigneron, L., Ruysschaert, J.-M. & Goormaghtigh, E. (1995) J. Biol. Chem. 270, 17685-17696] and on the gastric H+,K+-ATPase [Raussens, V., Ruysschaert, J.-M. & Goormaghtigh, E. (1997) J. Biol. Chem. 276, 262-270]. Extensive proteinase K proteolysis of open gastric tubulovesicles was performed here to generate the membrane-protected fragments of the H+,K+-ATPase. Secondary structure of the intact and of the membrane-protected segments was compared for oriented membrane films by attenuated total-reflection Fourier-transform infrared spectroscopy and by circular dichroism and for v...
Gastric H,K-ATPase topography: Amino acids 888–907 are cytoplasmic
Biochemical and Biophysical Research Communications, 1991
Gastric acidification is mediated by H,K-ATPase, an integral protein of apical membranes of gastric parietal cells. Hydropathy analysis of H,K-ATPase subunit primary structure predicts eight transmembrane (TM) domains, while omeprazole-binding data were interpreted in terms of ten TM domains (Mercier et al. (1991) FASEB J. 5, A749). In the present study, tryptic hydrolysis of gastric mucosal microsomes gave a set of peptides which bound the monoclonal antibody HK 12.18, a highly specific probe of the H,K-ATPase. An antiserum against the Cterminus of H,K-ATPase a subunit bound the same peptides, and one smaller peptide. The binding data suggested a putative epitepe for HK 12.18, and a 20-mer peptide encompassing this site was synthesized. This peptide bound directly to HK 12.18, displaced HK 12.18 from microsomal H,K-ATPase, and blocked HK 12.18 immunostaining of gastric parietal cells. In addition, intact gastric microsomes competitively inhibited binding of HK 12.18 to peptide-BSA conjugate. Taken together, these data place the HK 12.18 epitope between amino acids 888-907 and identify this domain as cytosolic. This result specifically excludes a pair of TM domains between the sixth and seventh TM a helices of the H,K-ATPase and supports a secondary structure model with eight TM domains.
Proton binding sites and conformational analysis of H+K+-ATPase
Biochemical and Biophysical Research Communications, 2005
It is proposed that the hydronium ion, H 3 O + , binds to the E1 conformation of the a-subunit of gastric proton pump. The H 3 O + binding cavities are characterized parametrically based on valence, sequence, geometry, and size considerations from comparative modeling. The cavities have scope for accommodating monovalent cations of different ionic radii. The H 3 O + transport is proposed to be aided by arenes which are arranged regularly along the pump starting from N-domain through the transmembrane region. Step-by-step structural changes accompanying H 3 O + occlusion are studied in detail. The observations corroborate well with earlier experimental studies.