Simulated docking and binding of Conantokin-P and Conantokin-Pr3to NMDA receptor GluN2B subunit: Roles of calcium ionsand gamma-carboxyglutamate residues

Abstract

Conantokin-P (con-P) and conantokin-Pr3 (con-Pr3), peptides isolated from Conus venoms have shown high potency and selectivity for the N-methyl-D-aspartate (NMDA) receptor GluN2B subunit. In this work, structural bases for these characteristics were investigated through in silico modelling and molecular dynamics simulations. Con-P and con-Pr3 were predicted to adopt stable α-helical conformations in the presence of Ca²⁺. The non-standard gamma-carboxyglutamate (Gla) residues in these conotoxins were observed to be important for the association of Ca²⁺. In con-P and con-Pr3, Gla residues form salt bridges with GluN2B basic amino acid residues. Additionally, basic amino acid residues of the conantokins form salt bridges with GluN2B acidic residues. Calcium binding is shown to enhance the participation of Gla residues in saltbridge formation of both con-P and con-Pr3. Molecular docking revealed the existence of con-P binding sites in both the external and internal faces of the GluN2B amino-terminal domain–agonist-binding domain (ATD-ABD) pocket. In contrast, con-Pr3 docked only to the internal face. For con-P, increased hydrogen bond interactions and more salt bridges were observed for docks at the external face compared to those at the internal face. Additionally, con-P binding at the external face of the ATD-ABD pocket can induce an inward stretching motion for the ATD. This stretching occurred with greater magnitude in the presence of Ca²⁺, suggesting that Ca²⁺ influenced conformational changes in the external face of the ATD-ABD pocket. Calcium ions also cause outward stretching for con-P, when bound at the internal face of the ATD-ABD pocket. Interestingly, for con-Pr3, Ca²⁺ resulted in minimal movement, while large outward stretching was observed in Ca²⁺-free conditions. Notably, the observed binding affinities for con-Pr3 reflect similar magnitudes as previous studies. Furthermore, molecular dynamic simulations revealed that con-P may form both dimers and trimers that are sustained by multiple Ca²⁺-Gla intermolecular bridges, while con-Pr3 may entail the recruitment of three sets of Gla residues to form a stable multimer. These characterizations of conotoxin binding to NMDA receptors provide bases for the development and testing of conantokin-based therapies for neurological conditions.