Abstract
The P-glycoprotein (P-gp, ABCB1) drug pump protects us from toxic compounds and confers multidrug resistance. Each of the homologous halves of P-gp is composed of a transmembrane domain (TMD) with 6 TM segments followed by a nucleotide-binding domain (NBD). The predicted drug- and ATP-binding sites reside at the interface between the TMDs and NBDs, respectively. Crystal structures and EM projection images suggest that the two halves of P-gp are separated by a central cavity that closes upon binding of nucleotide. Binding of drug substrates may induce further structural rearrangements because they stimulate ATPase activity. Here, we used disulfide cross-linking with short (8 Å) or long (22 Å) cross-linkers to identify domain-domain interactions that activate ATPase activity. It was found that cross-linking of cysteines that lie close to the LSGGQ (P517C) and Walker A (I1050C) sites of NBD1 and NBD2, respectively, as well as the cytoplasmic extensions of TM segments 3 (D177C or L175C) and 9 (N820C) with a short cross-linker activated ATPase activity over 10-fold. A pyrylium compound that inhibits ATPase activity blocked cross-linking at these sites. Cross-linking between the NBDs was not inhibited by tariquidar, a drug transport inhibitor that stimulates P-gp ATPase activity but is not transported. Cross-linking between extracellular cysteines (T333C/L975C) predicted to lock P-gp into a conformation that prevents close NBD association inhibited ATPase activity. The results suggest that trapping P-gp in a conformation in which the NBDs are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity.
Highlights
P-glycoprotein is an ATP-binding cassette (ABC) transporter that confers multidrug resistance
The results suggest that trapping P-gp in a conformation in which the nucleotide-binding domain (NBD) are closely associated likely mimics the structural rearrangements caused by binding of drug substrates that stimulate ATPase activity
We report that a pyrylium compound (P10) that inhibits ATPase activity blocked cross-linking between the NBDs while an inhibitor that is not transported [25] but stimulates ATPase activity did not inhibit cross-linking
Summary
P-glycoprotein is an ABC transporter that confers multidrug resistance. Results: Linkage of the nucleotide-binding domains with short but not long cross-linkers highly activated ATPase activity. In the mouse inward-facing mouse P-gp crystal structure, the cross-linkable cysteines would be predicted to be in close proximity (segments of the C-terminal ends were not resolved in the crystal structure) while the N-terminal and central regions that form the nucleotide-binding sandwich structure would be far apart These results showed that the C-terminal ends of the NBDs were not required to undergo significant separation during the catalytic cycle. Cross-linking with a short but not a long cross-linker was found to highly activate ATPase activity These results indicate that maintaining the NBD N-terminal and central regions in close proximity mimics activation of ATPase activity by covalent attachment of drug substrates to the drug-binding domain. We report that a pyrylium compound (P10) that inhibits ATPase activity blocked cross-linking between the NBDs while an inhibitor (tariquidar) that is not transported [25] but stimulates ATPase activity did not inhibit cross-linking
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