Human P-glycoprotein is active when the two halves are clamped together in the closed conformation.
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the two homologous halves of P-gp is composed of a transmembrane domain (TMD) with six TM segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures show drug pumps in the open and closed conformations, where the drug-binding pocket and NBDs are open or closed at the cytoplasmic side, respectively. Although it has been postulated that drug substrates enter the drug-binding pocket in the open conformation, it is unknown if they can enter in the closed conformation. To determine this, we introduced cysteines into regions of TM3 (residues 175-178) and TM9 (residues 820-822) that extend into the cytoplasm and are 4 A and 20 A apart in the closed and open conformations, respectively. The 12 double cysteine mutants were then cross-linked with a short cross-linker, M1M (4 A) at 0 degrees C to reduce thermal motion in the protein. Only mutant L175C/N820C was cross-linked. Cross-linking was not increased in the presence of ATP or drug substrates. Cross-linking increased its basal ATPase activity about 3-fold. Activity could be increased further by drug substrates such as verapamil and rhodamine B. These results suggest that P-gp in the membrane is in the closed conformation that has a high affinity for drug substrates.
Loo
Tip W
TW
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Bartlett
M Claire
MC
Clarke
David M
DM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D018432:Drug Resistance, Multiple; D006801:Humans; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases; D003545:Cysteine