Abstract
The P-glycoprotein (P-gp) drug pump (ABCB1) has two transmembrane domains and two nucleotide-binding domains (NBDs). Coupling of the drug-binding sites in the transmembrane domains to the NBDs occurs through interaction of the intracellular helices (IHs) with residues in the NBDs (IH1/IH4/NBD1 and IH2/IH3/NBD2). We showed previously that cross-linking of cysteines in IH3 and IH1 with a short cross-linker mimicked drug binding as it activated P-gp ATPase activity. Here we show that residue A259C(IH2) could be directly cross-linked to W803C(IH3). Cross-linking was inhibited by the presence of ATP and adenosine 5'-(β,γ-imino)triphosphate but not by ADP. Cross-linking of mutant A259C/W803C inhibited its verapamil-stimulated ATPase activity mutant, but activity was restored after addition of dithiothreitol. Because these residues are close to the ball-and-socket joint A266C(IH2)/Phe(1086)(NBD2), we mutated the adjacent Tyr(1087)(NBD2) close to IH3. Mutants Y1087A and Y1087L, but not Y1087F, were misprocessed, and all inhibited ATPase activity. Mutation of hydrophobic residues (F793A, L797A, L814A, and L818A) flanking IH3 also inhibited maturation. The results suggest that these residues, together with Trp(803) and Phe(804), form a large hydrophobic pocket. The results show that there is an important hydrophobic network at the IH2/IH3/NBD2 transmission interface that is critical for folding and activity of P-gp.
Highlights
Mutations to Hydrophobic Residues in IH3 and Flanking Regions Inhibit Maturation—According to the human P-gp model on the basis of the C. elegans structure, IH3 consists of residues Asp800 to Asp806 in ICL3 and is predicted to connect TMD2 to NBD2 (Fig. 1A) [17]
The first intracellular helices (IHs) (IH1 and IH3) in each transmembrane domain (TMD) (Fig. 1A) that link the TMDs to nucleotide-binding domains (NBDs) in the same half of the protein are generally only found in ABC exporters [46], whereas IH2 and IH4, which make the TMD1/NBD2 or TMD2/NBD1 connections, respectively (Fig. 1A), are found in all ABC transporters
For TMD2, five residues in IH3 interacted with NBD2, whereas 11 residues in IH4 make contacts with NBD1 [17]
Summary
It was predicted that the IHs of Sav1866 acted as interfaces to transmit conformational changes associated with ATP binding and hydrolysis at the NBDs to the TMDs [27]. In addition to acting as transmission interfaces, it is likely that the IHs play important roles in TMD/NBD interactions required for folding of P-gp because a truncation mutant lacking the NBDs will not mature in the absence of drug substrates [30]. It appears that NBD interactions with the TMDs are important for packing of the 12 TM segments during synthesis [21]. We tested whether cross-linking of IH3 to IH2 would have the opposite effect of cross-linking IH3 to IH1 [16] and cause inhibition, rather than stimulation, of ATPase activity
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