Effect of Methionine Oxidation and Substitution of α-Conotoxin TxID on α3β4 Nicotinic Acetylcholine Receptor

Abstract

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.

Degradation kinetics of α‐conotoxin TxID

“…10E, both TxID 0 and product II displayed similar inhibitory activities to native TxID. Though there was about 14-fold decrease in the IC 50 value of product I relative to native TxID, the result was similar to that reported previously [24]. Representative traces of these four peptides on a3b4 nAChR are shown in Fig.…”

“…Ren et al . used molecular dynamics simulations to show that the Met residue can establish a hydrophobic interaction with the residues in the binding pocket; when Met is oxidized, the hydrophobic interaction between the peptide and the receptor is weakened, resulting in decreased binding activity to α3β4 nAChR.…”

“…The side chain group of Met has a sulfur atom, which is susceptible to electron transfer oxidation to give sulfoxide . When Met was oxidized to form methionine sulfoxide in TxID, the inhibition on α3β4 nAChR decreased 13‐fold in potency compared to native TxID . Some researchers have suggested that adding antioxidants to react with oxidants could prevent degradation .…”

“…These results indicated that the cleavage of the amide bond in TxID has no effect on the activity of the a3b4 nAChR subtype, but the oxidation of Met affects the binding activity of this subtype. Ren et al [24] used molecular dynamics simulations to show that the Met residue can establish a hydrophobic interaction with the residues in the binding pocket; when Met is oxidized, the hydrophobic interaction between the peptide and the receptor is weakened, resulting in decreased binding activity to a3b4 nAChR.…”

Tethered peptide neurotoxins display two blocking mechanisms in the K + channel pore as do their untethered analogs

“…Therefore, neutralization of Lys 22 abolishes the voltage dependence of block in both cases, and the z of voltage-dependent inhibition was the same for T-HmK and HmK peptide, supporting a similar binding position for Lys 22 in the two toxin forms in the KcsA-Shaker pore. Because variants were expressed on the cell surface at similar levels as WT T-HmK, scanning with alanine point mutations provided a rapid and efficient process, avoiding the time and expense of producing peptides and the risks attendant with handling, such as difficulty of peptide synthesis and folding in vitro (32), residue modification such as methionine oxidation, and peptide degradation during storage (33). The result was a facile mapping of the seven most impactful residues in T-HmK on block of KcsA-Shaker, pointing to the binding interface upon the NMR-derived HmK structure.…”