µ-Conotoxins are VGSC blockers which are well studied and recently suggested for treatment of chronic pain. Here, we construct a new model of Nav1.4 and study the binding of µ-conotoxin; GIIIA to Nav1.4.
We use NavAb crystal structure (3RVY) as a template to create a model of Nav1.4 pore.
We align the critical (DEKA) residues forming the selectivity filter with the corresponding (EEEE) residues in NavAb. Sequences of the four domains of Nav1.4 are aligned with NavAb using ClustalW. A 3D model of the channel is created using Modeller. The Nav1.4 model is embedded in a lipid bilayer and simulated for 15 ns. The stable structure of Nav1.4 is used to create the Nav1.4-GIIIA complex. We use HADDOCK for docking and the best complex is selected for refinment via 20 ns MD simulations. From the trajectory data, we determine pair-residue interactions. According to the experimental data, R13, K16 and R19 are the most important residues for binding of GIIIA to Nav1.4. Our results show that R13 interacts with E403, E758 and D1532. K16 has stable and strong interaction with D1241 and R19 interacts with D762. This conformation is supported by several mutational data and rationalizes some of the experimental observations; For instance, the critical and unique role of R13 in pore blocking and the role of domain II in toxin binding.
To provide further validation for the complex, we calculate binding free energy from the potential of mean force (PMF) of GIIIA. The result reproduces the experimental value within 1 kcal/mol. In conclusion, the proposed complex of Nav1.4-GIIIA is in good agreement with several mutational data, and its accuracy is further supported by the binding free energy. These comparisons indicate that our model of VGSC pore is fairly accurate and provides a useful model for studying toxin interactions with VGSC channels.