Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter.
The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (V(i)) and fluoroaluminate (AlF(x)) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp-M2+-ADP-X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeF(x)) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and V(i), BeF(x) or AlF(x) using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with V(i) and ATP led to a 6-fold increase in the acrylamide quenching constant, K(SV), suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with V(i) and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)-ATP/TNP-ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeF(x)-trapped complex was much more polar than that of the V(i) and AlF(x) complexes. Functionally, all the trapped complexes were able to bind drugs and TNP-nucleotides with unchanged affinity compared with native Pgp.
Russell
Paula L
PL
Department of Molecular and Cellular Biology University of Guelph, Guelph, ON, Canada N1G 2W1.
Sharom
Frances J
FJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism*; D020106:Acrylamide / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002586:Cesium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005453:Fluorescence; D007455:Iodine / Q000378:metabolism; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D014301:Trinitrobenzenes / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020106:Acrylamide; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D002586:Cesium; D007455:Iodine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D014301:Trinitrobenzenes; D014364:Tryptophan