Identifying the lipid-protein interface and transmembrane structural transitions of the Torpedo Na,K-ATPase using hydrophobic photoreactive probes.
To identify regions of the Torpedo Na,K-ATPase alpha-subunit that interact with membrane lipid and to characterize conformationally dependent structural changes in the transmembrane domain, we have proteolytically mapped the sites of photoincorporation of the hydrophobic compounds 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) and the phosphatidylcholine analogue [(125)I]TIDPC/16. The principal sites of [(125)I]TIDPC/16 labeling were identified by amino-terminal sequence analysis of proteolytic fragments of the Na,K-ATPase alpha-subunit and are localized to hydrophobic segments M1, M3, M9, and M10. These membrane-spanning segments have the greatest levels of exposure to the lipid bilayer and constitute the bulk of the lipid-protein interface of the Na,K-ATPase alpha-subunit. The extent of [(125)I]TID and [(125)I]TIDPC/16 photoincorporation into these transmembrane segments was the same in the E(1) and E(2) conformations, indicating that lipid-exposed segments located at the periphery of the transmembrane complex do not undergo large-scale movements during the cation transport cycle. In contrast, for [(125)I]TID but not for [(125)I]TIDPC/16, there was enhanced photoincorporation in the E(2) conformation, and this component of labeling mapped to transmembrane segments M5 and M6. Conformationally sensitive [(125)I]TID photoincorporation into the M5 and M6 segments does not reflect a change in the levels of exposure of these segments to the lipid bilayer as evidenced by the lack of [(125)I]TIDPC/16 labeling of these two segments in either conformation. These results suggest that [(125)I]TID promises to be a useful tool for structural characterization of the cation translocation pathway and for conformationally dependent changes in the pathway. A model of the spatial organization of the transmembrane segments of the Na,K-ATPase alpha- and beta-subunits is presented.
Blanton
M P
MP
Departments of Pharmacology and Anesthesiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
McCardy
E A
EA
D000818:Animals; D001389:Azirines / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010713:Phosphatidylcholines / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D010777:Photochemistry; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014101:Torpedo
D001389:Azirines; D007457:Iodine Radioisotopes; D008563:Membrane Lipids; D008565:Membrane Proteins; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D019920:Photoaffinity Labels; C088423:1-palmitoyl-2-(decanedioyl mono-(2-iodo-4-(3-trifluoromethyl-3H-diazirin-3-yl)benzyl)ester)glycero-3-phosphocholine; C033174:3-(trifluoromethyl)-3-(3-iodophenyl)diazirine; D010450:Endopeptidases; D000254:Sodium-Potassium-Exchanging ATPase