Abstract
P-glycoprotein (Pgp) is an ATP hydrolysis driven multidrug efflux pump, which, when overexpressed in the plasma membrane of certain cancers, can lead to the failure of chemotherapy. Previously, we have presented a projection structure of nucleotide-free mouse Pgp from electron microscopic images of lipid monolayer-generated two-dimensional crystals ( Lee, J. Y., Urbatsch, I. L., Senior, A. E., and Wilkens, S. (2002) J. Biol. Chem. 277, 40125-40131 ). Here we have analyzed the structure of cysteine-free human Pgp from two-dimensional crystals that were generated with the same lipid-monolayer technique in the absence and presence of various nucleotides. The images show that human Pgp has a similar structure to the mouse protein. Furthermore, the analysis of projection structures obtained under different nucleotide conditions suggests that Pgp can exist in at least two major conformations, one of which shows a central cavity between the N- and C-terminal halves of the molecule and another in which the two halves have moved sideways, thereby closing the central cavity. Intermediate conformations were observed for some nucleotide/vanadate combinations. A low-resolution, three-dimensional model of human Pgp was calculated from tilted specimen crystallized in the presence of the non-hydrolyzable nucleotide analog, adenosine 5'-O-(thiotriphosphate). The structural analysis presented here adds to the emerging picture that multidrug ABC transporters function by switching between two major conformations in a nucleotide-dependent manner.
Highlights
Comparison of the Pgp Three-dimensional Model with the Crystal Structure of Sav1866—Because the q molecule seemed better resolved compared with the p molecule, we have focused on this three-dimensional map of Pgp for comparison with the three-dimensional crystal structure of the MgADP bound form of Staphylococcus aureus Sav1866 (Fig. 6, fitting of the p molecule is shown in supplemental Fig. S5) (Protein Data Bank (PDB) code 2hyd)
In an attempt to gain a better understanding of the conformational changes Pgp undergoes during ATP hydrolysis-driven drug translocation, we generated two-dimensional crystals of the nucleotide-free and nucleotide-bound protein for structural analysis by transmission electron microscopy
We continued to use the lipid bilayer-bound protein, because this is the physiological form of Pgp, and we utilized the human protein because of its optimal clinical relevance
Summary
Escherichia coli total lipids, and 1,2-dioleoyl-sn-glycero-3-[[N-(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl] (nickel salt) (Ni-NTA-DOGS) were obtained from Avanti Polar Lipids, Inc. n-Dodecyl--D-maltoside was obtained from Anatrace Inc. The contrast transfer function was corrected by inverting the phases between the first and second zero as implemented in IMAGIC 5. Correlation averaging starting from the first reference was iterated 2– 4 times using an average of the best subimages from the previous round until no further improvement was seen in the averages of the crystal patches. At this point, the motif was centered and the size of the reference was reduced to cover 16 (and later 9) unit cells, and correlation averaging was continued until stable results were obtained. Depending on the quality of the starting image (crystal size, crystalline order), final averages were calculated from 1000 to 2000 images. An inverse temperature factor B ϭ Ϫ2000 ÅϪ2 was imposed to compensate for the resolution dependent attenuation in the amplitudes
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