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 cr-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 hydrophobie compounds 3-(trifluoromethyl)3-(m-[I-125] [I-125]iodophenyl)diazirine ([I-125]TID) and the phosphatidylcholine analogue [I-125]TIDPC/16. The principal sites of [I-125]TIDPC/16 labeling were identifed by amino-terminal sequence analysis of proteolytic fragments of the Na,K-ATPase alpha -subunit and are localized to hydrophobic segments MI, 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 [I-125]TID and [125I]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 [I-125]- TID but not for [I-125]TIDPC/16, there was enhanced photoincorporation in the E2 conformation, and this component of labeling mapped to transmembrane segments M5 and MG. Conformationally sensitive [I-125]- TID photoincorporation into the M5 and MG segments does not reflect a change in the levels of exposure of these segments to the lipid bilayer as evidenced by the lack of [I-125]TIDPC/16 labeling of these two segments in either conformation. These results suggest that [125I]TID promises to he 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.