ABCB4 p.Phe947Ala
| Predicted by SNAP2: | A: N (61%), C: N (66%), D: D (85%), E: D (80%), G: D (71%), H: D (71%), I: N (82%), K: D (85%), L: N (57%), M: N (82%), N: D (75%), P: D (85%), Q: D (75%), R: D (85%), S: D (63%), T: N (57%), V: N (78%), W: D (71%), Y: D (71%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: N, Y: N, |
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[hide] Mutagenesis of transmembrane domain 11 of P-glycop... Biochemistry. 1996 Mar 19;35(11):3625-35. Hanna M, Brault M, Kwan T, Kast C, Gros P
Mutagenesis of transmembrane domain 11 of P-glycoprotein by alanine scanning.
Biochemistry. 1996 Mar 19;35(11):3625-35., 1996-03-19 [PMID:8639515]
Abstract [show]
The biochemical and genetic analyses of P-glycoprotein (P-gp) have indicated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport. Predicted transmembrane domain 11 (TM11) maps near a major drug binding site revealed by photoaffinity labeling, and mutations in this domain alter the substrate specificity of P-gp. To investigate further the role of TM11 in P-gp function in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of endogenous alanines. After transfection and overexpression in Chinese hamster ovary cells, pools of stable transfectants were analyzed for qualitative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by the wild-type protein. While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of activity against one of the four drugs tested. Although the phenotype of individual mutants was varied, replacements at most mutation-sensitive positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp. When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutral mutations was not random but segregated with a more hydrophobic (mutation-insensitive) face and a more hydrophilic (mutation-sensitive) face of a putative amphipathic helix. The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more hydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp. Finally, the conservation of the two residues most sensitive to mutations (Y949 and Y953) in TM11, and in the homologous TM5, of all mammalian P-gps and also in other ABC transporters, suggests that these residues and domains may play an important role in structural as well as mechanistic aspects common to this family of proteins.
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No. Sentence Comment
156 These two mutations are also predicted to map near the top half of TM11, within the outer lipid leaflet of Table 1: Drug Survival Characteristics of Mass Populations Cells Stably Transfected with Wild-Type or Mutant mdr3 cDNAsa mdr3 ACT ADR COL VBL V933A 32 ( 3 (17×) 370 ( 200 (19×) 590 ( 180 (21×) 200 ( 20 (24×) F934A 37 ( 2 (19×) 110 ( 40 (6×) 240 ( 80 (9×) 200 ( 30 (24×) G935A 26 ( 3 (14×) 200 ( 110 (10×) 350 ( 20 (13×) 120 ( 20 (14×) I936A 23 ( 5 (12×) 100 ( 40 (5×) 170 ( 40 (6×) 80 ( 13 (10×) T937A 50 ( 10 (27×) 280 ( 110 (15×) 620 ( 100 (23×) 160 ( 6 (20×) F938A 42 ( 6 (22×) 140 ( 60 (7×) 360 ( 80 (13×) 260 ( 20 (32×) S939A 46 ( 1 (24×) 160 ( 80 (8×) 280 ( 30 (10×) 160 ( 30 (20×) F940A 43 ( 4 (22×) 380 ( 130 (20×) 980 ( 210 (36×) 240 ( 30 (29×) T941A 48 ( 3 (25×) 100 ( 40 (5×) 140 ( 20 (5×) 130 ( 40 (16×) Q942A 60 ( 6 (32×) 100 ( 40 (5×) 190 ( 10 (7×) 270 ( 70 (33×) A943G 41 ( 4 (22×) 90 ( 10 (5×) 350 ( 60 (13×) 380 ( 40 (46×) M944A 29 ( 6 (15×) 450 ( 190 (23×) 730 ( 90 (27×) 200 ( 40 (24×) M945A 56 ( 2 (29×) 180 ( 50 (9×) 340 ( 80 (13×) 210 ( 50 (26×) Y946A 16 ( 2 (8×) 310 ( 160 (16×) 350 ( 30 (13×) 100 ( 8 (13×) F947A 32 ( 2 (17×) 310 ( 100 (16×) 660 ( 120 (24×) 170 ( 30 (20×) S948A 25 ( 3 (13×) 210 ( 80 (11×) 370 ( 60 (13×) 210 ( 40 (26×) Y949A 18 ( 2 (9×) 60 ( 20 (3×) 180 ( 20 (6×) 190 ( 15 (23×) A950G 46 ( 6 (24×) 280 ( 70 (15×) 520 ( 120 (19×) 200 ( 30 (25×) A951G 60 ( 10 (32×) 300 ( 30 (16×) 560 ( 120 (21×) 280 ( 30 (34×) C952A 24 ( 5 (12×) 360 ( 180 (19×) 500 ( 50 (18×) 140 ( 50 (18×) F953A 8 ( 1 (4×) 28 ( 7 (1×) 45 ( 9 (2×) 100 ( 13 (13×) WT 50 ( 10 (25×) 290 ( 80 (15×) 450 ( 100 (16×) 210 ( 40 (25×) LR73 2 ( 0.1 (1×) 19 ( 6 (1×) 27 ( 4 (1×) 8 ( 2 (1×) a The drug survival of Chinese hamster ovary drug-sensitive cells (LR73) and of cell clones transfected with either wild-type or mutant mdr3 is expressed as the D50 (in nanograms per milliliter), or the dose necessary to reduce the plating efficiency of the control and transfected cells by 50%.
X
ABCB4 p.Phe947Ala 8639515:156:1418
status: NEW124 Eight of the mutations (V933A, T937A, F940A, M944A, M945A, F947A, A950G, and A951G) had no significant effect on function, and the corresponding mutant P-gps expressed levels of resistance to each drug similar to that of WT.
X
ABCB4 p.Phe947Ala 8639515:124:59
status: NEW135 Strikingly, seven of the eight mutations which were without consequences on mdr3 function map to the highly hydrophobic face of this helix, with six of them (V933A, T937A, F940A, M944A, F947A, and A951G) clustered in a continuous mutation-insensitive segment (Figure 4).
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ABCB4 p.Phe947Ala 8639515:135:186
status: NEW136 Included in this group are two nonconservative substitutions, F940A and F947A, in which a large aromatic side chain is replaced by a methyl group, and the T937A mutant, in which a hydrophilic hydroxyl group is eliminated.
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ABCB4 p.Phe947Ala 8639515:136:72
status: NEW