ABCMdb

PMID: 12609990

Loo TW, Bartlett MC, Clarke DM
Substrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding.
J Biol Chem. 2003 Apr 18;278(16):13603-6. Epub 2003 Feb 27., 2003-04-18 [PubMed]

Sentences

No. Mutations Sentence Comment

30 ABCB1 p.Pro350Cys These concentrations gave maximal stimulation of the mutant P-gp mutant P350C. Login to comment 51 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys These mutants contained P350C(TM6) and another cysteine on the cytoplasmic half of TMs 7-12 (i.e. P350C ϩ another cysteine at positions: 711-723 (TM7), 770 -783 (TM8), 828 - 840 (TM9), 867-879 (TM10), 935-947 (TM11), and 986-994 (TM12)) or S993C(TM12) with another cysteine in the cytoplasmic half of TMs 1-6 (i.e. S993C ϩ another cysteine at positions: 51-61 (TM1), 130-141 (TM2), 185-196 (TM3), 226-237 (TM4), 293-304 (TM5), and 343-351 (TM6)). Login to comment 70 ABCB1 p.Pro350Cys An example was mutant P350C- (TM6)/G939C(TM11). Login to comment 85 ABCB1 p.Val991Cys Membranes prepared from HEK 293 cells expressing mutants P350C(TM6)/ A935C(TM11), P350C(TM6)/G939C(TM11), or P350C(TM6)/V991C- (TM12) were preincubated for 10 min at 21 °C with no drug, 1 mM progesterone, 0.1 mM cyclosporin A, 5 mM colchicine, or 1 mM demecolcine. Login to comment 91 ABCB1 p.Gly939Cys In mutant P350C(TM6)/G939C- (TM11), progesterone was more efficient in promoting cross-linking than cyclosporin A. Login to comment 92 ABCB1 p.Val991Cys In mutant P350C(TM6)/V991C- (TM12), the drug substrates colchicine, demecolcine, and progesterone were equally effective in promoting cross-linking. Login to comment 107 ABCB1 p.Ala935Cys ABCB1 p.Val991Cys Accordingly, we tested whether the drug substrates colchicine, demecolcine, progesterone, cis-(Z)-flupenthixol, verapamil, or vinblastine stimulated the ATPase activities of histidine-tagged mutants P350C(TM6)/A935C- (TM11), P350C(TM6)/G939C(TM11), and P350C(TM6)/V991C- (TM12). Login to comment 108 ABCB1 p.Pro350Cys Their activities were compared with that of mutant P350C (parent). Login to comment 109 ABCB1 p.Pro350Cys We previously showed that mutant P350C exhibited high levels of drug-stimulated ATPase activity (11). Login to comment 119 ABCB1 p.Pro350Cys The activities of the mutant P-gps are expressed relative to mutant P350C. Login to comment 123 ABCB1 p.Ser993Cys In the absence of drug substrate, disulfide cross-linking occurs between Pro350 (yellow ball) and S993C (purple ball). Login to comment 124 ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys ABCB1 p.Ala935Cys ABCB1 p.Val991Cys In the presence of drug substrate (progesterone), TM segments 11 and 12 undergo rotational and/or lateral movements so that cross-linking can occur between P350C and V991C (black ball) in TM12 and with A935C (red ball) and G939C (turquoise ball) in TM11. Login to comment 133 ABCB1 p.Pro350Cys Substrate-induced Cross-linking between TM Segments of P-gp atUniversityofNorthCarolinaatChapelHill,onSeptember29,2011www.jbc.orgDownloadedfrom substrates such as progesterone are effective in promoting new cross-links with P350C (Fig. 2). Login to comment 134 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys In the absence of drug substrate, residue P350C in TM6 can be cross-linked to S993C in TM12. Login to comment 135 ABCB1 p.Gly939Cys ABCB1 p.Ala935Cys ABCB1 p.Val991Cys The presence of progesterone, however, promoted cross-linking of residue P350C(TM6) with two residues in TM 11 (A935C and G939C) and to residue V991C in TM12. Login to comment 139 ABCB1 p.Pro350Cys ABCB1 p.Val991Cys Therefore, the presence of drug substrate (progesterone) likely causes a slight rotation/rearrangement of TM12 relative to TM6 such that residue V991C comes closer to P350C. Login to comment 142 ABCB1 p.Gly939Cys When the residues in TM11 are modeled as an ␣-helical wheel, residues A935 and G939C are found on the same face of the TM segment (Fig. 5B). Login to comment 144 ABCB1 p.Gly939Cys ABCB1 p.Pro350Cys Cyclosporin A also promoted cross-linking between TM6 and TM11, but only between P350C and G939C. Login to comment 145 ABCB1 p.Pro350Cys ABCB1 p.Ala935Cys It is possible that cyclosporin A altered the tilt or distance between TM6 and TM11 such that only A939C but not A935C were close enough to P350C to be cross-linked. Login to comment