ABCC8 p.Asp18Ala
| Predicted by SNAP2: | A: N (61%), C: N (72%), E: N (87%), F: D (63%), G: N (57%), H: N (53%), I: D (59%), K: N (72%), L: D (59%), M: D (53%), N: N (87%), P: D (63%), Q: N (72%), R: N (57%), S: N (87%), T: N (93%), V: N (61%), W: D (80%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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[hide] Delineation of the functional site of alpha-dendro... J Biol Chem. 1998 Sep 25;273(39):25393-403. Gasparini S, Danse JM, Lecoq A, Pinkasfeld S, Zinn-Justin S, Young LC, de Medeiros CC, Rowan EG, Harvey AL, Menez A
Delineation of the functional site of alpha-dendrotoxin. The functional topographies of dendrotoxins are different but share a conserved core with those of other Kv1 potassium channel-blocking toxins.
J Biol Chem. 1998 Sep 25;273(39):25393-403., [PMID:9738007]
Abstract [show]
We identified the residues that are important for the binding of alpha-dendrotoxin (alphaDTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys5 and Leu9 decreased affinity more than 1000-fold, and substitutions of Arg3, Arg4, Leu6, and Ile8 by 5-30-fold. Substitution of Lys5 by norleucine or ornithine also greatly altered the binding properties of alphaDTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type alphaDTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser38 and Arg46 also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Thus, the functional site of alphaDTX includes six major binding residues, all located in its N-terminal region, with Lys5 and Leu9 being the most important. Comparison of the functional site of alphaDTX with that of DTX-K, another dendrotoxin (Smith, L. A., Reid, P. F., Wang, F. C., Parcej, D. N., Schmidt, J. J., Olson, M. A., and Dolly, J. O. (1997) Biochemistry 36, 7690-7696), reveals that they only share the predominant lysine and probably a leucine residue; the additional functional residues differ from one toxin to the other. Comparison of the functional site of alphaDTX with those of structurally unrelated potassium channel-blocking toxins from venomous invertebrates revealed the common presence of a protruding key lysine with a close important hydrophobic residue (Leu, Tyr, or Phe) and few additional residues. Therefore, irrespective of their phylogenetic origin, all of these toxins may have undergone a functional convergence. The functional site of alphaDTX is topographically unrelated to the "antiprotease site" of the structurally analogous bovine pancreatic trypsin inhibitor.
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No. Sentence Comment
134 The other substitutions (P2A, H10A, R11A, R15A, Y17A, and D18A) and the previously described mutation D12N (32) did not significantly reduce the affinity.
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ABCC8 p.Asp18Ala 9738007:134:58
status: NEW193 The spectra were recorded at 20 °C in 0.1-cm cuvettes, with a protein concentration of 5-10 M. TABLE I Affinity of ␣DTX analogs determined by their ability to compete with [125 I]␣DTX for binding to rat brain synaptosomal membranes ␣DTX Ki a Ki analog/Ki WTb pM WT 4 Ϯ 1 1 P2A 10.6 Ϯ 1.1 2.9 Ϯ 1.0 R3A 28.5 Ϯ 4.6 7.9 Ϯ 3.1 R4A 38 Ϯ 12 11 Ϯ 5.75 K5A 5800 Ϯ 700 1594 Ϯ 574 K5Orn 471 Ϯ 42 128 Ϯ 43 K5Nle 5700 Ϯ 400 1547 Ϯ 487 L6A 43.4 Ϯ 12 12.4 Ϯ 6.1 I8A 118 Ϯ 26 33.2 Ϯ 14.8 L9A 4160 Ϯ 900 1170 Ϯ 518 H10A 1.6 Ϯ 0.2 0.45 Ϯ 0.15 R11A 3.0 Ϯ 0.1 0.8 Ϯ 0.2 R15A 4.6 Ϯ 0.1 1.22 Ϯ 0.33 Y17A 3.7 Ϯ 0.1 1.0 Ϯ 0.27 D18A 3.4 Ϯ 0.1 0.91 Ϯ 0.25 K19A 2.3 Ϯ 0.1 0.63 Ϯ 0.17 Q27A 2.9 Ϯ 0.1 0.78 Ϯ 0.21 Q31A 3.65 Ϯ 0.05 0.98 Ϯ 0.26 E33A 5.3 Ϯ 0.05 1.42 Ϯ 0.37 R34A 7.1 Ϯ 0.1 1.9 Ϯ 0.5 D36A 4.85 Ϯ 0.1 1.30 Ϯ 0.35 S38A 23.5 Ϯ 6.5 6.64 Ϯ 3.24 S44A 6.7 Ϯ 0.1 1.8 Ϯ 0.45 R46A 562 Ϯ 152 160 Ϯ 78 K48A 2.8 Ϯ 0.05 0.75 Ϯ 0.2 E51A 3.65 Ϯ 0.05 0.98 Ϯ 0.26 R54A 5.4 Ϯ 0.05 1.44 Ϯ 0.38 R55A 4.0 Ϯ 0.21 1.08 Ϯ 0.3 I58A 3.45 Ϯ 0.25 1.27 Ϯ 0.63 a Ki Ϯ S.E. values are calculated from competition experiments as described in the legend of Fig. 5. b WT, wild type.
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ABCC8 p.Asp18Ala 9738007:193:800
status: NEW