ABCMdb

ABCB1 p.Trp232Arg

Predicted by SNAP2:	A: D (53%), C: N (72%), D: D (75%), E: D (71%), F: N (66%), G: D (59%), H: D (53%), I: N (57%), K: D (75%), L: N (57%), M: N (82%), N: D (66%), P: D (85%), Q: D (66%), R: D (75%), S: D (53%), T: D (53%), V: N (57%), Y: N (72%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, Y: D,

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[hide] The W232R suppressor mutation promotes maturation ... Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11. Loo TW, Bartlett MC, Clarke DM
The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants.
Biochemistry. 2011 Feb 8;50(5):672-85. Epub 2011 Jan 11., 2011-02-08 [PMID:21182301]

Abstract [show]
ATP-binding cassette (ABC) proteins contain two nucleotide-binding domains (NBDs) and two transmembrane (TM) domains (TMDs). Interdomain interactions and packing of the TM segments are critical for function, and disruption by genetic mutations contributes to disease. P-glycoprotein (P-gp) is a useful model to identify mechanisms that repair processing defects because numerous arginine suppressor mutations have been identified in the TM segments. Here, we tested the prediction that a mechanism of arginine rescue was to promote intradomain interactions between TM segments and restore interdomain assembly. We found that suppressor W232R(TM4/TMD1) rescued mutants with processing mutations in any domain and restored defective NBD1-NBD2, NBD1-TMD2, and TMD1-TMD2 interactions. W232R also promoted packing of the TM segments because it rescued a truncation mutant lacking both NBDs. The mechanism of W232R rescue likely involved intradomain hydrogen bond interactions with Asn296(TM5) since only N296A abolished rescue by W232R and rescue was only observed when Trp232 was replaced with hydrogen-bonding residues. In TMD2, suppressor T945R(TM11) also promoted packing of the TM segments because it rescued the truncation mutant lacking the NBDs and suppressed formation of alternative topologies. We propose that T945R rescue was mediated by interactions with Glu875(TM10) since T945E/E875R promoted maturation while T945R/E875A did not.

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No. Sentence Comment
48 The W232R mutation may stabilize TMD1 as it promotes maturation of a mutant lacking the critical NBD2-TMD1 interface (ΔNBD2- P-gp) (42). Login to comment
49 Whether the W232R mutation could promote maturation of a TMD1 þ 2 mutant lacking both NBDs is unknown. Login to comment
54 FIGURE 1: Rescue of P-gp mutants containing processing mutations in different domains by W232R. Login to comment
58 (B) HEK 293 cells were transfected with A52-tagged wild-type P-gp, mutants G251V, G251V/W232R, and G251V/ W232A,orplasmidvector(control).Wholecellextractsweresubjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
59 (C) HEK 293 cells were transfected with A52-tagged mutants containing processing mutations in different domains and with or without the W232R mutation ((ΔY490(NBD1) ( W232R, P709A(linker region) ( W232R, G722A(TMD2) ( W232R, or L1260A(NBD2) ( W232R). Login to comment
84 Baby hamster kidney (BHK) cells expressing A52-tagged wild-type P-gp or mutants G251V, G251V/W232R, W232R, W232A, N296A, E875A, or T945A were generated as described previously (25). Login to comment
91 The G251V or L1260A processing mutations were introduced into Cys-less P-gp containing pairs of cysteines in different domains (L443C(NBD1)/ S909C(TMD2), L531C(NBD1)/C1074(NBD2), or C137(TMD1)/A935C(TMD2)) with or without the W232R mutation. Login to comment
92 The L1260A and L1260A/W232R mutations were also introduced into a Cys-less P-gp containing the A266C(TMD1)/ F1086C(NBD2) mutations. Login to comment
99 Membranes were prepared from HEK 293 cells expressing A52-tagged TMD1 þ 2 or TMD1 þ 2(W232R) and suspended in TBS, pH 7.4, at a protein concentration of 5 mg/mL. Login to comment
111 RESULTS W232R Promotes Maturation of Mutants with Processing Mutations in Any Domain. Login to comment
112 The W232R suppressor mutation that promotes maturation of the G251V processing mutant is located in TM4 of TMD1 (Figure 1A). Login to comment
116 It was found that only the W232R change promoted maturation of the mutant (Figure 1B, lanes 1-3). Login to comment
117 The amount of mature P-gp relative to total (mature plus immature) was 70 ( 6% for mutant W232R/G251V and 92 ( 5% for wild-type P-gp. Login to comment
123 We previously observed that W232R could rescue the ΔY490 (NBD1) and G251V (TMD1) P-gp processing mutants (42). Login to comment
124 We then tested whether W232R could rescue mutants containing processing mutations in other domains. Login to comment
125 The W232R mutation was introduced into mutants P709A (linker region), G722A (TMD2), or L1260A (NBD2). Login to comment
127 The previously reported ΔY490 and ΔY490/W232R mutants (42) were included for comparison. Login to comment
128 In all cases, introduction of the W232R mutation rescued the processing mutants such that the 170 kDa mature form of P-gp was the major product (Figure 1C). Login to comment
129 The results show that the W232R mutation could rescue mutants with processing mutations in any domain. Login to comment
135 It is possible that the W232R mutation promotes maturation because the charged arginine helps TM4 to insert stably into the membrane. Login to comment
144 Indeed, all of the mutations except W232R (Figure 2B, lane 12) inhibited maturation when expression was carried out in the absence of drug substrates (Figure 2C). Login to comment
146 The results show that only the W232R mutation promoted maturation in the absence of cyclosporin A. Login to comment
152 Effect of W232R on Domain-Domain Interactions. Login to comment
155 To test if the W232R mutation restored domain-domain contacts, it was introduced into the G251V or L1260A processing mutants that also contained pairs of cysteines at the TMD1-TMD2 (C137(TMD1)/A935C(TMD2), NBD1-NBD2 (L531C(NBD1)/C1074(NBD2), or NBD1-TMD2 (L443C(NBD1)/S909C(TMD2) interfaces. Login to comment
156 The G251V and L1260A parents were used because the G251V mutation is in the same domain as W232R (TMD1) whereas L1260A is in another domain (NBD2). Login to comment
158 Introduction of the W232R mutation into all of the G251V (Figure 3A, lanes 3, 7, and 11) or L1260A (Figure 3B, lanes 3, 7, and 11) double-cysteine processing mutants restored maturation. Login to comment
159 In the absence of the W232R mutation, all of the double cysteine mutants yielded immature protein that did not show cross-linking when treated with the copper phenanthroline (CuP) oxidant (lanes 2, 6, and 10 in Figure 3A,B). Login to comment
160 The mature W232R mutants, however, showed cross-linking between the cysteines when they were treated with oxidant (lanes 4, 8, and 12 in Figure 3A,B). Login to comment
161 The results suggest that the W232R mutation promotes maturation by inducing long-range conformation changes to promote TMD1-TMD2, NBD1-NBD2, and NBD1-TMD2 interactions in P-gp. Login to comment
162 Since the L1260A mutation is located in the NBD2 (Figure 1A), we also examined the effects of W232R on NBD2-TMD1 interactions. Login to comment
164 Accordingly, we introduced the W232R mutation into mutant A266C/F1086C/L1260A. Login to comment
166 Figure 3C shows that the W232R mutation promoted maturation of the mutant A266C/F1086C/L1260A (lane 3) and that only the mature protein was cross-linked (lane 4). Login to comment
167 The N296A Mutation Abolishes W232R Rescue. Login to comment
177 The model in Figure 1A, however, suggests that W232R must enhance matura- tionbya different mechanismsince itdoes not lie closetoany TM segments in TMD2. Login to comment
179 Therefore, W232R may promote maturation through hydrogen bond interactions with residues in TM5 or TM6. Login to comment
181 To test for evidence of hydrogen bond interactions of W232R with residues in TM segments 5, 6, or 12, potential hydrogen bond partners (Thr294(TM5), Asn296(TM5), Ser298(TM5), Ser344(TM6), Gln347(TM6), Ser349(TM6), Ser351(TM6)), (Gln990(TM12), Ser992(TM12), or Ser993(TM12)) (see Figure 4A,B) were mutated to alanine in a G251V/W232R background. Login to comment
182 The rationale was that removal of a hydrogen-bonding partner would abolish the ability of W232R to promote maturation. Login to comment
184 It was observed that only one mutation in TM5 (N296A) inhibited maturation of the G251V/W232R mutant when it was expressed in the absence of cyclosporin A (Figure 4C, lane 7). Login to comment
185 By contrast, mutation of potential hydrogen-bonding residues in TM6 (Ser344, Q347, S349, or Q351) or TM12 (Gln990, Ser992, or S993) to alanine did not affect maturation of G251V/W232R (Figure 4D). Login to comment
186 Introduction of the N296A mutation into G251V/W232R(TM4) caused the mutant to behave in a fashion similar to the original FIGURE 3: Effect of W232R on cross-linking between domains of processing mutants. Login to comment
187 Membranes were prepared from cells expressing P-gp processing mutants G251V ( W232R (A) or L1260A ( W232R (B) that also contained pairs of cysteines in various domains (L443C(NBD1)/S909C(TMD2), L531C(NBD1)/C1074(NBD2), C137(TMD1)/A935C(TMD2)). Login to comment
188 Membranes were also prepared from cells expressing the L1260A processing mutant ( W232R containing cysteines in TMD1 and NBD2 (A266C/F1086C) (C). Login to comment
192 FIGURE 4: Effect of mutating residues capable of forming hydrogen bonds with W232R on maturation of G251V. Login to comment
194 (C) HEK 293 cells transfected with A52-tagged mutant G251V or G251V/ W232R containing alanine mutations in potential hydrogen-bonding residues in TM5 (T294A, N296A, S298A) or G251V/N296A were expressed in the absence (-) or presence (þ) of cyclosporin A (cyclo A). Login to comment
200 Like the G251V parent (Figure 4C, lanes 1 and 2), the G251V/W232R/N296A mutant (Figure 4C, lane 7) showed about 15% maturation efficiency when expressed in the absence of drug substrates, and the mature 170 kDa P-gp was the major product when expressed in the presence of cyclosporin A (Figure 4C, lane 8). Login to comment
201 Therefore, the presence of the N296A mutation prevented the ability of W232R to promote maturation of the G251V mutant. Login to comment
203 These results suggest that Arg232(TM4) promotes maturation of the G251V mutant through hydrogen bond interactions with Asn296(TM5) because the N296A mutation inhibited the ability of W232R to promote maturation of G251V (Figure 4C, lane 7). Login to comment
205 If W232R rescues by forming a hydrogen bond with Asn296, then we infer from the models (Figure 4A,B) that it would require a slight rotation of TM5 to bring the Asn side chain closer to the side chainof arginine at position 232. Login to comment
212 Rescue by W232R Does Not Require the NBDs. Login to comment
215 Alanine-scanning mutagenesis of potential hydrogen bond partners suggested that W232R rescue involved Asn296 in TM5 (Figure 4C, lane 7). Login to comment
216 Since residues W232R and Asn296 were located in the TMDs, it was possible that the NBDs would not be required for rescue. Login to comment
217 To test if the NBDs were required for rescue, the W232R mutation was introduced into a truncation mutant lacking both NBDs (TMD1 þ 2(W232R)). Login to comment
218 When TMD1 þ 2(W232R) was expressed in HEK 293 cells, it was found that about 50% of the protein was present as mature protein (Figure 6A). Login to comment
219 No detectable mature protein was observed in cells expressing TMD1 þ 2 lacking W232R or with mutant TMD1 þ 2(W232R/N296A) (Figure 6A). Login to comment
222 To test if W232R also converted TMD1 þ 2 into a protease-resistant conformation, membranes prepared from cells expressing TMD1 þ 2 with or without W232R were treated with various concentrations of trypsin. Login to comment
228 FIGURE 6: Effect of the W232R mutation on maturation and protease sensitivity of a P-gp truncation mutant lacking the NBDs (TMD1 þ 2). Login to comment
229 (A) Whole cell extracts of cells transfected with A52-tagged wild-type, W232R, or W232R/N296A forms of TMD1 þ 2 were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
230 (B) Membranes prepared from cells expressing A52-tagged wild-type (-W232R) or mutant W232R (þW232R) forms of TMD1 þ 2 were treated with the indicated concentrations of trypsin and samples subjected to immunoblot analysis. Login to comment
232 mature form of TMD1 þ 2(W232R) was 15.5 times more resistant totrypsinrelativetotheimmatureformsoftheprotein(Figure6B). Login to comment
233 Rescue by W232R resembled drug rescue as both converted TMD1 þ 2 to a mature protease-resistant conformation. Login to comment
270 To test if rescue by an arginine suppressor mutation yielded an active transporter, we examined the ability of mutants G251V and G251V/W232R to confer resistance to cytotoxic compounds colchicine, paclitaxel, and vinblastine. Login to comment
272 Stable BHK cell lines expressing wild-type P-gp or mutants G251V, W232R, or G251V/W232R were generated by cotransfecting P-gp cDNAs with pWL-neo vector followed by selection with G418. Login to comment
277 The W232R mutation altered the drug resistance profile as it conferred only a 4-fold increase in resistance to vinblastine but showed relatively high levels of resistance to colchicine (16-fold; P < 0.05) and paclitaxel (19-fold; P < 0.05) (Figure 9A). Login to comment
278 The decreased resistance to vinblastine and increased resistance to colchicine were consistent with the reported effects of W232R on drug-stimulatedATPaseactivity (42).MutantW232Rshowed an increased S50 (concentration required for 50% stimulation of activity) for vinblastine and increased maximum stimulation in the presence of colchicine. Login to comment
281 Introduction of the W232R mutation into G251V, however, yielded a functional P-gp. Login to comment
282 Cells expressing mutant G251V/W232R were more resistant to colchicine (12-fold; P < 0.05) compared to cells expressing wild-type P-gp (9-fold) but were less resistant to vinblastine (3-fold (P < 0.05) versus 17-fold) and paclitaxel (18-fold (P < 0.05) versus 29-fold). Login to comment
283 These results show that the W232R mutation restored maturation of mutant G251V P-gp to yield a functional transporter at the cell surface. Login to comment
297 (A) Stable BHK cell lines expressing no P-gp (control), equivalent levels of wild type, mutants G251V, W232R, or G251V/W232R P-gp were incubated for 6 days in the presence of various concentrations of drug substrates vinblastine (gray bars), colchicine (white bars), or paclitaxel (black bars). Login to comment
311 The large effect of the T945A mutation on ATPase activity is consistent with the observation that wild-type P-gp containing W232R exhibits verapamiland rhodamine-stimulated ATPase activity whereas wild-type P-gp containing T945R does not (42). Login to comment
313 First, the W232R suppressor mutation located inthe first domain (TMD1) can repair defects in maturation caused by processing mutations located in any other domain. Login to comment
316 Second, the NBDs are not required for rescue by W232R or T945R. Login to comment
350 P-gp TMD1 þ 2 could be rescued to yield mature protein in a protease-resistant conformation by the W232R mutation. Login to comment
352 Therefore, one possible explanation for the ability of the W232R- (TM4) mutation to promote maturation of the P-gp processing mutants was that the positively charged side chain of arginine could serve as a better anchor for TM4. Login to comment
362 Multiple topologies have been detected (34-37), and drug substrates (8) or suppressor mutations like W232R (this study) can convert the domains from protease-sensitive to protease- resistantconformations.TheplasticityoftheTMDsmaycontribute to alterations in the substrate specificity of P-gp since they contain the drug-binding sites. Login to comment

[hide] A salt bridge in intracellular loop 2 is essential... Biochemistry. 2013 May 14;52(19):3194-6. doi: 10.1021/bi400425k. Epub 2013 May 3. 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., [PMID:23634976]

Abstract [show]
There is no high-resolution structure of the human P-glycoprotein (P-gp, ABCB1) drug pump. Homology models based on the crystal structures of mouse and Caenorhabditis elegans P-gps show extensive contacts between intracellular loop 2 (ICL2, in the first transmembrane domain) and the second nucleotide-binding domain. Human P-gp modeled on these P-gp structures yields different ICL2 structures. Only the model based on the C. elegans P-gp structure predicts the presence of a salt bridge. We show that the Glu256-Arg276 salt bridge was critical for P-gp folding.

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Sentences [show]
No. Sentence Comment
59 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