Secondary structure of P-glycoprotein investigated by circular dichroism and amino acid sequence analysis.
P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp.
Dong
M
M
Institut de Biologie et Chimie des Protéines, UPR 412 CNRS, 7 Passage du Vercors F-69367, Lyon Cedex 07, France.
Ladavière
L
L
Penin
F
F
Deléage
G
G
Baggetto
L G
LG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D006528:Carcinoma, Hepatocellular; D002942:Circular Dichroism; D006031:Glycosylation; D006801:Humans; D007945:Leukemia, Lymphoid; D051379:Mice; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D051381:Rats; D017421:Sequence Analysis; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1