ABCMdb

ABCB1 p.Tyr953Phe

Predicted by SNAP2:	A: D (66%), C: D (59%), D: D (75%), E: D (75%), F: N (53%), G: D (71%), H: D (53%), I: N (53%), K: D (85%), L: D (59%), M: D (71%), N: N (57%), P: D (91%), Q: D (71%), R: D (85%), S: D (59%), T: D (66%), V: D (53%), W: D (71%),
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, W: D,

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Publications
[hide] Arginines in the first transmembrane segment promo... J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2. Loo TW, Bartlett MC, Clarke DM
Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11.
J Biol Chem. 2008 Sep 5;283(36):24860-70. Epub 2008 Jul 2., 2008-09-05 [PMID:18596043]

Abstract [show]
A key goal is to correct defective folding of mutant ATP binding cassette (ABC) transporters, as they cause diseases such as cystic fibrosis. P-glycoprotein (ABCB1) is a useful model system because introduction of an arginine at position 65 of the first transmembrane (TM) segment could repair folding defects. To determine the mechanism of arginine rescue, we first tested the effects of introducing arginines at other positions in TM1 (residues 52-72) of a P-glycoprotein processing mutant (G251V) that is defective in folding and trafficking to the cell surface (20% maturation efficiency). We found that arginines introduced into one face of the TM1 helix (positions 52, 55, 56, 59, 60, 62, 63, 66, and 67) inhibited maturation, whereas arginines on the opposite face of the helix promoted (positions 64, 65, 68, and 71) or had little effect (positions 61, and 69) on maturation. Arginines at positions 61, 64, 65, and 68 appeared to lie close to the drug binding sites as they reduced the apparent affinity for drug substrates such as vinblastine and verapamil. Therefore, arginines that promoted maturation may face an aqueous drug translocation pathway, whereas those that inhibited maturation may face the lipid bilayer. The highest maturation efficiencies (60-85%) were observed with the Arg-65 and Arg-68 mutants. Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Therefore, arginine may rescue defective folding by promoting packing of the TM segments through hydrogen bond interactions.

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Sentences [show]
No. Sentence Comment
7 Mutations that removed hydrogen bond acceptors (Y950F/Y950A or Y953F/Y953A) in TM11 predicted to lie close to Arg-65 or Arg-68 inhibited maturation but did not affect maturation of the G251V parent. Login to comment
228 Conservative replacement of Tyr-950 or Tyr-953 with Phe would only disrupt hydrogen bond interactions. Login to comment
229 Mutants L65R/G251V/Y950F, M68R/G251V/ Y950A/Y953A, and M68R/G251V/Y950F/Y953F were constructed, and the cDNAs were expressed in HEK 293 cells. Login to comment
231 Immunoblot analysis shows the Y950A/Y953A and Y950F/Y953F changes in M68R/G251V (Fig. 8A, lanes 11 and 12) or Y950F change in L65R/G251V (Fig. 8B, lane 5) reduced maturation to levels similar to that observed in the G251V parent. Login to comment
232 Introduction of the Y950F and Y953F FIGURE 7. Login to comment
308 This possibility is supported by the observations that the conservative Y950F and Y953F mutations reduced maturation of the M68R/G251V mutant to levels observed with the G251V parent. Login to comment
309 An arginine introduced at position 65 in TM1 only appeared to form a hydrogen bond with Tyr-950 as the Y950F mutation but not the Y953F change reduced maturation of the L65R/G251V mutant to levels observed with the G251V parent. Login to comment

[hide] Pore-exposed tyrosine residues of P-glycoprotein a... Mol Pharmacol. 2014 Mar;85(3):420-8. doi: 10.1124/mol.113.088526. Epub 2013 Dec 23. Donmez Cakil Y, Khunweeraphong N, Parveen Z, Schmid D, Artaker M, Ecker GF, Sitte HH, Pusch O, Stockner T, Chiba P
Pore-exposed tyrosine residues of P-glycoprotein are important hydrogen-bonding partners for drugs.
Mol Pharmacol. 2014 Mar;85(3):420-8. doi: 10.1124/mol.113.088526. Epub 2013 Dec 23., [PMID:24366667]

Abstract [show]
The multispecific efflux transporter, P-glycoprotein, plays an important role in drug disposition. Substrate translocation occurs along the interface of its transmembrane domains. The rotational C2 symmetry of ATP-binding cassette transporters implies the existence of two symmetry-related sets of substrate-interacting amino acids. These sets are identical in homodimeric transporters, and remain evolutionary related in full transporters, such as P-glycoprotein, in which substrates bind preferentially, but nonexclusively, to one of two binding sites. We explored the role of pore-exposed tyrosines for hydrogen-bonding interactions with propafenone type ligands in their preferred binding site 2. Tyrosine 953 is shown to form hydrogen bonds not only with propafenone analogs, but also with the preferred site 1 substrate rhodamine123. Furthermore, an accessory role of tyrosine 950 for binding of selected propafenone analogs is demonstrated. The present study demonstrates the importance of domain interface tyrosine residues for interaction of small molecules with P-glycoprotein.

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Sentences [show]
No. Sentence Comment
53 Construction of P-gp Mutants The following primers were used for generation of the Y307F, Y310F, Y307F/Y310F, Y950F, Y953F, and Y950F/Y953F mutations of hexa-His-tagged human P-gp in the entry vector pENTR4: Y307F- forward, 59-CTTTCCTGCTGATCTTTGCATCTTATGCTCTGGCC-39; Y307F-reverse, 59-GGCCAGAGCATAAGATGCAAAGATCAGCAGG AAAG-39; Y310F-forward, 59-CTTTCCTGCTGATCTATGCATCTTT TGCTCTGGCC-39; Y310F-reverse, 59-GGCCAGAGCAAAAGATGCA- TAGATCAGCAGGAAAG-39; Y307F/Y310F-forward, 59-CTTTCCTG CTGATCTTTGCATCTTTTGCTCTGGCC-39; Y307F/Y310F-reverse, 59-GGCCAGAGCAAAAGATGCAAAGATCAGCAGGAAAG-39; Y950F- forward, 59-TCACCCAGGCAATGATGTTTTTTTCCTATGCTGGATG- 39; Y950F-reverse, 59-CATCCAGCATAGGAAAAAAACATCATTGCC TGGGTGA-39; Y953F-forward, 59-ACCCAGGCAATGATGTATTTTT CCTTTGCTGGATGTTTC-39; Y953F-reverse, 59-GAAACATCCAGCAAA GGAAAAATACATCATTGCCTGGGT-39; Y950F/Y953F-forward, 59-CCTT CACCCAGGCAATGATGTTTTTTTCCTTTGCTGGATGTTTCC -39; and Y950F/Y953F-reverse, 59-GGAAACATCCAGCAAAGGAAAAAAACATCA TTGCCTGGGTGAAGG-39. Login to comment
117 Mutant Y950F showed a similar transport rate as wild-type protein, whereas mutant Y953F showed a significantly decreased rate (54% 6 12% of wild-type) (Fig. 2). Login to comment
118 The double mutant Y950F/Y953F showed a decrease that was comparable with that observed for the Y953F single mutant alone. Login to comment
121 The effect of the Y953F mutation on rh123 efflux should be abolished by introducing selector residue R132 and should be more pronounced when deselecting site 1 by introducing selector residue R773. Login to comment
125 This is illustrated by comparable transport activity of the Q132R/Y950F, Q132R/Y953F, and Q132R/ Y950F/Y953F mutants. Login to comment
128 By contrast, transport rates were found to be lower in the Q773R/Y953F mutant compared with the Q773R mutant alone, although this decrease did not reach statistical significance. Login to comment
137 Q773R/Y950F/Y953F was expressed at the surface, but no rh123 efflux was detectable. Login to comment
146 The wild-type showed an IC50 value of 518 6 141 nM, whereas the values for the single mutants Y950F and Y953F were 1348 6 229 and 1207 6 362 nM, respectively. Login to comment
148 An analogous pattern was seen for GPV031 (wild-type, 85 6 22 nM; Y950F, 238 6 84 nM; Y953F, 287 6 109 nM; and Y950F/Y953F, 630 6 87 nM) (Fig. 3C). Login to comment
152 In contrast with protonatable propafenones, a higher IC50 value was observed for the Y953F, but not for the Y950F, single mutant. Login to comment
153 The fold change for the double mutant was comparable with that observed for the Y953F single mutant (wild-type, 2239 6 391 nM; Y950F, 2984 6 79 nM, n.s.; Y953F, 15,946 6 2941 nM, 7.1-fold change relative to wild-type; and Y950F/Y953F, 17,819 6 2106 nM, 8-fold change). Login to comment
159 (A) Open diamonds, wild-type; filled circles, Y950F/Y953F; triangles, Y950F; squares, Y953F. Login to comment
164 However, IC50 values were still higher in the Q132R/Y953F mutation. Login to comment
168 For the nonprotonatable acid amide GPV366, a higher IC50 value was observed in the Q132R/Y953F mutant (Q132R, 1074 6 282 nM; and Q132R/Y953F, 3261 6 965 nM), whereas the Y950F mutation in the same background did not affect potency (966 6 259 nM) (Fig. 4C). Login to comment
171 The higher IC50 values in the Q132R/Y950F/Y953F mutant were not due to an additive effect of the two tyrosine mutations on binding of propafenones. Login to comment
173 Certainly, the effect is not brought about by a global perturbance of protein structure and function, because the Q132R mutant and the Q132R/Y950F/Y953F triple mutant showed comparable rh123 transport rates (Fig. 2). Login to comment
193 Identical transport rates were found for the Q132R and the Y953F mutants in the Q132R background. Login to comment
198 As expected, the double mutant showed an IC50 value that was comparable with that of the Y953F mutant. Login to comment
202 However, higher IC50 values were found for the Y953F mutant, although less pronounced than in the wild-type background. Login to comment
212 For GPV005, mutant Q132R/Y953F showed a 1.5-fold higher IC50 value than the Q132R mutation, whereas the fold changes were 2.1-fold for GPV031 and 3.0-fold for uncharged GPV366. Login to comment
217 The triple Q132R/Y950F/Y953F mutant showed a further increase in IC50 values for all compounds, which is obviously not due to interaction with tyrosines. Login to comment