ABCC8 p.Tyr179Phe
| Predicted by SNAP2: | A: D (59%), C: D (63%), D: D (59%), E: D (75%), F: D (66%), G: N (53%), H: D (63%), I: D (71%), K: D (71%), L: D (71%), M: D (71%), N: N (61%), P: D (75%), Q: D (63%), R: D (80%), S: N (53%), T: D (59%), V: D (66%), W: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: N, 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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[hide] Structural model of ligand-G protein-coupled recep... J Biol Chem. 2011 Sep 9;286(36):31661-75. Epub 2011 Jun 17. Marquer C, Fruchart-Gaillard C, Letellier G, Marcon E, Mourier G, Zinn-Justin S, Menez A, Servent D, Gilquin B
Structural model of ligand-G protein-coupled receptor (GPCR) complex based on experimental double mutant cycle data: MT7 snake toxin bound to dimeric hM1 muscarinic receptor.
J Biol Chem. 2011 Sep 9;286(36):31661-75. Epub 2011 Jun 17., [PMID:21685390]
Abstract [show]
The snake toxin MT7 is a potent and specific allosteric modulator of the human M1 muscarinic receptor (hM1). We previously characterized by mutagenesis experiments the functional determinants of the MT7-hM1 receptor interaction (Fruchart-Gaillard, C., Mourier, G., Marquer, C., Stura, E., Birdsall, N. J., and Servent, D. (2008) Mol. Pharmacol. 74, 1554-1563) and more recently collected evidence indicating that MT7 may bind to a dimeric form of hM1 (Marquer, C., Fruchart-Gaillard, C., Mourier, G., Grandjean, O., Girard, E., le Maire, M., Brown, S., and Servent, D. (2010) Biol. Cell 102, 409-420). To structurally characterize the MT7-hM1 complex, we adopted a strategy combining double mutant cycle experiments and molecular modeling calculations. First, thirty-three ligand-receptor proximities were identified from the analysis of sixty-one double mutant binding affinities. Several toxin residues that are more than 25 A apart still contact the same residues on the receptor. As a consequence, attempts to satisfy all the restraints by docking the toxin onto a single receptor failed. The toxin was then positioned onto two receptors during five independent flexible docking simulations. The different possible ligand and receptor extracellular loop conformations were described by performing simulations in explicit solvent. All the docking calculations converged to the same conformation of the MT7-hM1 dimer complex, satisfying the experimental restraints and in which (i) the toxin interacts with the extracellular side of the receptor, (ii) the tips of MT7 loops II and III contact one hM1 protomer, whereas the tip of loop I binds to the other protomer, and (iii) the hM1 dimeric interface involves the transmembrane helices TM6 and TM7. These results structurally support the high affinity and selectivity of the MT7-hM1 interaction and highlight the atypical mode of interaction of this allosteric ligand on its G protein-coupled receptor target.
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No. Sentence Comment
156 hM1 mutant Region pKi NMS pKX MT7 KX(mut)/KX(WT) WT 10.22 Ϯ 0.04 10.47 Ϯ 0.04 H90A E1 10.20 Ϯ 0.09 10.66 Ϯ 0.20 0.6 W91A E1 9.23 ؎ 0.33 9.30 ؎ 0.06 14.9 W101A TM3 9.29 Ϯ 0.02 9.79 Ϯ 0.13 4.8 Y166A E2 9.83 Ϯ 0.03 10.33 Ϯ 0.32 1.4 L167F E2 9.93 Ϯ 0.02 10.48 Ϯ 0.11 1.0 E170A E2 10.20 Ϯ 0.04 10.20 Ϯ 0.07 1.9 E170K E2 10.16 Ϯ 0.02 9.31 ؎ 0.11 14.6 R171A E2 9.76 Ϯ 0.01 9.50 ؎ 0.29 9.4 L174A E2 10.21 Ϯ 0.08 10.51 Ϯ 0.01 0.9 L174P E2 10.15 Ϯ 0.01 9.37 ؎ 0.14 12.5 Q177E E2 10.08 Ϯ 0.04 10.58 Ϯ 0.17 0.8 Y179F E2 9.85 Ϯ 0.02 9.25 ؎ 0.18 16.5 L183A E2 10.00 Ϯ 0.11 10.44 Ϯ 0.20 1.1 Q185A E2 10.18 Ϯ 0.10 10.59 Ϯ 0.37 0.8 K392A E3 10.20 Ϯ 0.12 10.57 Ϯ 0.18 0.8 D393A E3 9.96 Ϯ 0.09 10.50 Ϯ 0.23 0.9 E397A E3 10.21 Ϯ 0.06 10.54 Ϯ 0.20 0.9 W400A TM7 10.12 Ϯ 0.04 9.43 ؎ 0.02 11.0 E401A TM7 9.76 Ϯ 0.02 10.20 Ϯ 0.10 1.9 TABLE 2 Affinity constants and variations in free energy of interaction of wild-type and modified MT7 toxins for wild-type and mutated hM1 receptors Data were inferred from binding experiments with ͓3 H͔NMS and calculated with the ternary complex equation.
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ABCC8 p.Tyr179Phe 21685390:156:643
status: NEW160 hM1 receptor hM1 region MT7 toxin MT7 loop pKX ⌬⌬Gint kcal/mol WT WT 10.47 Ϯ 0.04 H90A E1 WT 10.66 Ϯ 0.20 W10A 1 11.62 Ϯ 0.28 -2.08 Ϯ 0.40 Y30A 2 9.55 Ϯ 0.28 ؉1.14 Ϯ 0.37 Y51A 3 10.42 Ϯ 0.13 ϩ0.20 Ϯ 0.11 R52A 3 10.42 Ϯ 0.44 -0.04 Ϯ 0.59 W91A E1 WT 9.30 Ϯ 0.06 W10A 1 7.69 Ϯ 0.05 ؉1.09 Ϯ 0.07 Y30A 2 7.81 Ϯ 0.07 ؉1.64 Ϯ 0.09 Y51A 3 8.79 Ϯ 0.17 ϩ0.44 Ϯ 0.24 R52A 3 8.54 Ϯ 0.13 ϩ0.66 Ϯ 0.17 W101A TM3 WT 9.79 Ϯ 0.13 W10A 1 8.57 Ϯ 0.14 ϩ0.57 Ϯ 0.19 Y30A 2 9.08 Ϯ 0.09 ϩ0.60 Ϯ 0.11 R34A 2 7.74 Ϯ 0.10 -0.37 Ϯ 0.21 R52A 3 9.05 Ϯ 0.19 ϩ0.62 Ϯ 0.25 E170A E2 WT 10.20 Ϯ 0.07 W10A 1 8.42 Ϯ 0.20 ؉1.33 Ϯ 0.25 Y30A 2 8.84 Ϯ 0.28 ؉1.47 Ϯ 0.38 S32A 2 8.63 Ϯ 0.16 ؉1.43 Ϯ 0.22 R34A 2 7.50 Ϯ 0.36 ϩ0.60 Ϯ 0.49 M35A 2 8.63 Ϯ 0.13 ؉0.87 Ϯ 0.17 Y36A 2 7.97 Ϯ 0.13 ؉1.24 Ϯ 0.17 Y51A 3 9.71 Ϯ 0.31 ϩ0.41 Ϯ 0.42 R52A 3 8.57 Ϯ 0.18 ؉1.77 Ϯ 0.27 R171A E2 WT 9.50 Ϯ 0.29 K5A 1 9.25 Ϯ 0.18 -0.10 Ϯ 0.24 W10A 1 7.76 Ϯ 0.18 ؉1.26 Ϯ 0.24 Y30A 2 8.22 Ϯ 0.14 ؉1.49 Ϯ 0.43 S32A 2 8.46 Ϯ 0.08 ؉0.72 Ϯ 0.11 M35A 2 7.68 Ϯ 0.46 ؉1.20 Ϯ 0.62 Y36A 2 7.03 Ϯ 0.21 ؉1.55 Ϯ 0.28 K48A 2 9.48 Ϯ 0.02 -0.13 Ϯ 0.02 Y51A 3 8.67 Ϯ 0.12 ؉0.89 Ϯ 0.16 R52A 3 8.35 Ϯ 0.17 ؉1.17 Ϯ 0.23 L174A E2 WT 10.51 Ϯ 0.01 W10A 1 9.19 Ϯ 0.20 ؉0.71 ؎ 0.27 Y30A 2 9.45 Ϯ 0.17 ؉1.08 Ϯ 0.23 S32A 2 9.66 Ϯ 0.20 ϩ0.48 Ϯ 0.27 R34A 2 7.52 Ϯ 0.13 ؉1.19 Ϯ 0.42 M35A 2 8.72 Ϯ 0.10 ؉1.16 Ϯ 0.13 Y36A 2 8.59 Ϯ 0.23 ؉0.83 Ϯ 0.31 Y51A 3 10.06 Ϯ 0.23 ϩ0.37 Ϯ 0.31 R52A 3 9.23 Ϯ 0.22 ؉1.35 Ϯ 0.29 Y179F E2 WT 9.25 Ϯ 0.18 K5A 1 9.15 Ϯ 0.15 -0.28 Ϯ 0.20 W10A 1 7.62 Ϯ 0.19 ؉1.13 Ϯ 0.25 R34A 2 Ͻ6 > ؉1.35 K48A 2 9.21 Ϯ 0.01 -0.09 Ϯ 0.01 R52A 3 Ͻ6 >؉4.00 K392A E3 WT 10.57 Ϯ 0.18 W10A 1 9.88 Ϯ 0.22 -0.13 Ϯ 0.29 F11A 1 10.19 Ϯ 0.20 ϩ0.27 Ϯ 0.27 D393A E3 WT 10.50 Ϯ 0.23 S8A 1 10.05 Ϯ 0.06 ϩ0.21 Ϯ 0.08 W10A 1 10.10 Ϯ 0.02 -0.53 Ϯ 0.02 F11A 1 10.21 Ϯ 0.24 ϩ0.35 Ϯ 0.09 Y51A 3 10.00 Ϯ 0.34 ϩ0.42 Ϯ 0.46 E397A E3 WT 10.54 Ϯ 0.20 W10A 1 8.64 Ϯ 0.22 ؉1.58 Ϯ 0.31 F11A 1 9.89 Ϯ 0.21 ϩ0.64 Ϯ 0.28 W400A TM7 WT 9.43 Ϯ 0.02 K5A 1 9.60 Ϯ 0.05 -0.66 Ϯ 0.07 W10A 1 7.61 Ϯ 0.32 ؉1.38 Ϯ 0.44 F11A 1 8.29 Ϯ 0.25 ؉1.29 Ϯ 0.33 Y30A 2 8.49 Ϯ 0.08 ؉0.90 Ϯ 0.10 S32A 2 8.64 Ϯ 0.15 ϩ0.38 Ϯ 0.20 R34A 2 Ͻ6 >؉1.58 M35A 2 8.52 Ϯ 0.13 -0.11 Ϯ 0.12 Y36A 2 7.37 Ϯ 0.29 ؉1.00 Ϯ 0.38 K48A 3 9.41 Ϯ 0.06 -0.12 Ϯ 0.07 R52A 3 7.73 Ϯ 0.08 ؉0.91 Ϯ 0.17 E401A TM7 WT 10.20 Ϯ 0.10 R34A 2 7.41 Ϯ 0.37 ϩ0.61 Ϯ 0.45 Modeling of MT7-Dimeric hM1 Muscarinic Receptor Complex 31666 account this flexibility, we generated open conformations of loop E2 by molecular dynamics.
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ABCC8 p.Tyr179Phe 21685390:160:2107
status: NEW[hide] The M1 muscarinic receptor allosteric agonists AC-... Mol Pharmacol. 2010 Oct;78(4):648-57. Epub 2010 Jul 21. Jacobson MA, Kreatsoulas C, Pascarella DM, O'Brien JA, Sur C
The M1 muscarinic receptor allosteric agonists AC-42 and 1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one bind to a unique site distinct from the acetylcholine orthosteric site.
Mol Pharmacol. 2010 Oct;78(4):648-57. Epub 2010 Jul 21., [PMID:20660086]
Abstract [show]
Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one) , and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol 61:1297-1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.
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No. Sentence Comment
167 In comparison, the affinity of N-desmethylclozapine was only slightly changed on W101A and W101F, where a 2-fold decrease and 1.25-fold increase in potency were measured, respectively. Substitution of Tyr179 with either alanine or phenylalanine resulted in only minor changes in functional potencies -10 -9 -8 -7 -6 -5 -4 -3 0 20 40 60 80 100 Ile Phe Log [Agonist] (M) %Max -10 -9 -8 -7 -6 -5 -4 -3 0 20 40 60 80 100 Asn Gly Log [Agonist] (M) %Max -10 -9 -8 -7 -6 -5 -4 -3 0 20 40 60 80 100 Asn Thr Log [Agonist] (M) %Max Ach AC42 TBPB Fig. 4.
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ABCC8 p.Tyr179Phe 20660086:167:201
status: NEW173 The affinity of acetylcholine was decreased by only 2.5and 2-fold on Y179A and Y179F, respectively.
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ABCC8 p.Tyr179Phe 20660086:173:79
status: NEW176 In comparison, AC-42 exhibited a 2.8-fold decrease in affinity on Y179F, whereas the affinity for TBPB was unchanged compared with wild-type M1 receptors.
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ABCC8 p.Tyr179Phe 20660086:176:66
status: NEW