ABCMdb

PMID: 23634976

Loo TW, Clarke DM
A salt bridge in intracellular loop 2 is essential for folding of human p-glycoprotein.
Biochemistry. 2013 May 14;52(19):3194-6. doi: 10.1021/bi400425k. Epub 2013 May 3., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCB1 p.Ala266Cys ABCB1 p.Phe1086Cys ABCB1 p.Phe1086Cys ICL2 appears to play a key role in coupling NBD1-TMD2 interactions because cysteines introduced into ICL2 (A266C) and NBD2 (F1086C) could be cross-linked and the F1086C change abolished activity.13 While both structures predict that residues 261-267 form an interhelical loop (IH2) (forms the ball portion of the ball-and-socket ICL2-NBD connection), adjacent amino acids were predicted to adopt loop or b1;-helical structures in the mouse or C. elegans structures, respectively. Login to comment 27 ABCB1 p.Glu256Ala ABCB1 p.Arg276Ala By contrast, none of the Gly mutations in ICL1, -3, or -4 inhibited maturation.10 To test if the charged amino acids Glu256 and Arg276 were important for maturation, we characterized mutants E256A and R276A. Login to comment 28 ABCB1 p.Glu256Ala ABCB1 p.Arg276Ala Figure 1E shows that E256A or R276A mutations inhibited maturation. Login to comment 29 ABCB1 p.Glu256Ala ABCB1 p.Arg276Ala While the mature 170 kDa protein was the major product of wild-type P-gp, the immature 150 kDa protein was the major product of mutants E256A and R276A. Login to comment 31 ABCB1 p.Glu256Ala ABCB1 p.Arg276Ala ABCB1 p.Glu275Ala ABCB1 p.Glu273Ala ABCB1 p.Lys271Ala ABCB1 p.Lys272Ala ABCB1 p.Glu255Ala It was found that the E255A, K271A, K272A, E273A, and E275A mutants were different from the E256A and R276A mutants because they yielded mature 170 kDa P-gp as their major product (Figure 1E). Login to comment 33 ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg If a salt bridge between Glu256 and Arg276 were important for the maturation of human P-gp, it would be expected that an E256R/R276E mutant would still mature. Login to comment 34 ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg Accordingly, we reversed the positions of the glutamic acid and arginine residues (E256R/R276E) and tested the mutant`s ability to undergo maturation. Login to comment 35 ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg The mature 170 kDa P-gp protein was the major product in mutant E256R/R276E (Figure 2A). Login to comment 37 ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg The E256R or R276E mutation, however, inhibited maturation of P-gp (Figure 2A). Login to comment 38 ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg For example, Figure 2B shows that the 150 kDa E256R protein was sensitive to treatment with endoglycosidase H or PNGase F. Similar results were obtained when mutant R276E was treated with endoglycosidase H or PNGase F (data not shown). Login to comment 40 ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg To test if mutant E256R/R276E yielded an active protein, the histidine-tagged mutant was expressed in HEK293 cells, isolated by nickel chelate chromatography, and assayed for verapamil-stimulated ATPase activity. Login to comment 41 ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg Verapamil was used because it is transported by P-gp14 and strongly stimulates (>10-fold) the ATPase activity of human P-gp.15 The E256R/ R276E mutant showed verapamil-stimulated ATPase activity that was similar to that of the wild-type protein (Figure 2C). Login to comment 44 ABCB1 p.Glu256Ala ABCB1 p.Arg276Ala To address this question, we first tested whether it was possible to promote maturation of the E256A or R276A mutant. Login to comment 45 ABCB1 p.Glu256Ala ABCB1 p.Arg276Ala We previously showed that P-gp was inactive when it was trapped in the ER as a core-glycosylated intermediate.16 Drug substrates (e.g., cyclosporine A) can promote maturation of P-gp processing mutants.17 Mutants E256A and R276A were expressed in HEK293 cells in the presence or absence of 10 bc;M cyclosporine A. Login to comment 48 ABCB1 p.Glu256Ala ABCB1 p.Arg276Ala The putative Glu256-Arg276 salt bridge was not essential for activity as both the E256A and R276A mutants showed wild-type verapamil-stimulated ATPase activity (Figure S1B of the Supporting Information). Login to comment 59 ABCB1 p.Trp232Arg In support of this hypothesis, we previously reported that a W232R mutation in TM4 appears to act as a suppressor mutation to rescue P-gp processing mutants through hydrogen bond interactions with Asn296 in TM5.18 Processing mutations likely trap polytopic membrane proteins like P-gp and CFTR in protease-sensitive conformations with incomplete packing of the TM segments.19,20 Suppressor mutations in the TMDs can Figure 2. Login to comment 61 ABCB1 p.Arg276Glu ABCB1 p.Arg276Glu ABCB1 p.Glu256Arg ABCB1 p.Glu256Arg (A) Whole cell sodium dodecyl sulfate (SDS) extracts of wild-type P-gp and mutants E256R, R276E, and E256R/R276E. Login to comment