Abstract
Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer’s activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.
Highlights
Covalent linkage of cysteine residues by disulfide bonds is fundamental for the folding, stability, and function of many peptides and proteins [1,2,3,4]
Interactions between the isomers and the channel protein were examined via molecular experiments for their potency to block the human skeletal muscle voltage-gated sodium channel present study, these isomers and further analogs
This study provides conceptual framework to understand with the pore of how different disulfide connectivities affect the structure of μ-PIIIA
Summary
Covalent linkage of cysteine residues by disulfide bonds is fundamental for the folding, stability, and function of many peptides and proteins [1,2,3,4]. It was was shown that thestructural biological therapeutic potential and they are models to study the impact of disulfide bonds on the structural activities and receptor specificities of some μ-, μO-, and α-conotoxins changed significantly if the the stability of disulfide-rich peptides and proteins. 3-disulfide-bonded μ-conotoxins antagonize voltage-gated sodium channels μ-Conotoxins are so-called pore blockers because they bind to the extracellular pore vestibule and, native disulfide patterns theμ-conotoxins peptides were experimentally modified [9,10,11,12]. Interactions between the isomers and the channel protein were examined via molecular experiments for their potency to block the human skeletal muscle voltage-gated sodium channel present study, these isomers and further analogs were analyzed in electrophysiological examined via molecular docking simulations
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