Nucleotide-induced structural changes in P-glycoprotein observed by electron microscopy.
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.
Lee
Jyh-Yeuan
JY
Department of Biochemistry, University of California, Riverside, California 92521, USA.
Urbatsch
Ina L
IL
Senior
Alan E
AE
Wilkens
Stephan
S
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D018432:Drug Resistance, Multiple / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D008567:Membranes, Artificial; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013873:Thionucleotides / Q000737:chemistry* / Q000378:metabolism; D014638:Vanadates / Q000737:chemistry* / Q000378:metabolism
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008567:Membranes, Artificial; D013873:Thionucleotides; D014638:Vanadates; C022570:adenosine 5'-O-(2-thiotriphosphate); D000255:Adenosine Triphosphate