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Mar Drugs
2018 Jun 20;166:. doi: 10.3390/md16060215.
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Effect of Methionine Oxidation and Substitution of α-Conotoxin TxID on α3β4 Nicotinic Acetylcholine Receptor.
Ren J, Li R, Ning J, Zhu X, Zhangsun D, Wu Y, Luo S.
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α-Conotoxin TxID was discovered from Conus textile by gene cloning, which has 4/6 inter-cysteine loop spacing and selectively inhibits α3β4 nicotinic acetylcholine receptor (nAChR) subtype. However, TxID is susceptible to modification due to it containing a methionine (Met) residue that easily forms methionine sulfoxide (MetO) in oxidative environment. In this study, we investigated how Met-11 and its derivatives affect the activity of TxID using a combination of electrophysiological recordings and molecular modelling. The results showed most TxID analogues had substantially decreased activities on α3β4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on α3β4 nAChR. However, one mutant, [M11I]TxID, displayed potent inhibition at α3β4 nAChR with an IC50 of 69 nM, which only exhibited 3.8-fold less compared with TxID. Molecular dynamics simulations were performed to expound the decrease in the affinity for α3β4 nAChR. The results indicate replacement of Met with a hydrophobic moderate-sized Ile in TxID is an alternative strategy to reduce the impact of Met oxidation, which may help to redesign conotoxins containing methionine residue.
Figure 1. Sequences alignment of α-CTx TxID analogues with Cys (I–III, II–IV) disulfide bond connectivity (A) and structures of TxID (B) and [MO]TxID (C). (A) The sequences of TxID analogues are shown and the substituted residues at position 11 are highlighted in red. The # indicates an amidated C terminus; (B) Structural representation of TxID (PDB ID code 2M3I); (C) Spatial structure of [MO]TxID while Met residue is oxidized to MetO by addition of oxygen to its sulfur atom, which was produced by PyMOL.
Figure 2. HPLC chromatograms and mass spectra of α-CTx TxID and [MO]TxID. Peptides were analyzed on a reversed phase analytical Vydac C18 (5 μm, 4.6 mm × 250 mm) HPLC column using a linear gradient of a 10–40% buffer B, and 90–60% buffer A over 20 min, where B = 0.05% TFA in 90% ACN; A = 0.075% TFA in water. The elution profile was monitored by measuring the absorbance at 214 nm. (A) HPLC chromatogram of α-CTx TxID; (B) Electrospray ionization mass spectrometry (ESI-MS) data for TxID with observed monoisotopic mass of 1488.56 Da; (C) HPLC chromatogram of [MO]TxID; (D) ESI-MS data for [MO]TxID with an observed monoisotopic mass of 1504.56 Da.
Figure 3. α3β4 nAChR-inhibitory activities of TxID and [MO]TxID. α3β4 nAChR was expressed in Xenopus oocytes as described under “Materials and Methods.†(A) TxID at 1 μM was added to the chamber and incubated with oocyte for 5 min. Then TxID was washed out and the response to 1-s pulses to ACh was again measured (arrow). “C†indicates control responses to Ach; (B) Representative ACh-evoked currents of rat α3β4 nAChR obtained in presence of [MO]TxID at 1 μM; (C) Representative ACh-evoked currents of rat α3β4 nAChR obtained that the oocyte was exposed to 100 nM TxID; (D) A representative response of one typical oocyte was exposed to 100 nM [MO]TxID; (E) Concentration-response analysis of the activities of TxID and [MO]TxID on rat α3β4 nAChR subtype. Error bars denote the means ± SEM. of the data from 3 to 8 separate oocytes.
Figure 4. Inhibition of α3β4 nAChR by TxID and its Met substitution analogues. (A) Bar graph of normalized inhibition of ACh-evoked current is generated by methionine-substituted TxID analogues and native TxID. All peptides were tested at 1 μM and data are represented as mean ± SEM (n = 3–6); (B) Concentration-response curves are obtained for inhibition of α3β4 nAChR. Error bars denote the means ± SEM of the data from 3 to 8 separate oocytes.
Figure 5. Molecular interactions between peptides and α3β4 nAChR through homology modeling and MD simulation. The α3 subunit is drawn in green, the β4 is in cyan, and the peptides are in brown. Amino acids around 4 Å radius of the Met and its substitutions are labeled. (A) The molecular model was shown between TxID and α3β4 nAChR during 50 ns MD simulations; (B) Snapshot at 50 ns of [MO]TxID and α3β4 interface; (C) Snapshot at 50 ns of [M11I]TxID and α3β4 interface.
Figure 6. MD simulations demonstrate the distance difference between rat β4-I78 and Met-11 or Ile-11. (A) For native TxID, the distance between the sulfur atom of Met-11 and side-chain Cδ of β4-I78 lies within 3.5 Å; (B) For analogue [M11I]TxID, the distance between Cγ2 of Ile-11 and side-chain Cδ of β4-I78 is 4.6 Å.
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