From simulation to application: Elucidating Protoxin-2 binding mechanism in pain channel Nav1.7 through dynamics simulation and biophysics.

Abstract

Human Nav1.7 is a subtype of voltage-gated sodium channel that initiates and propagates action potentials during pain signaling. It functions by amplifying pain signals during tissue damage, making it an excellent pharmacological target for designing novel analgesics. Protoxin-2 is a known gating modifier toxin that targets the voltage-sensing domain (VSD) in human Nav1.7. However, the difficulty in tuning Protoxin-2 subtype selectivity remains a bottleneck in maximizing its pharmacologic potential. Such complexity in targeting Nav1.7 with Protoxin-2 arises mainly due to the limited knowledge of the biophysical properties and structural basis of toxin-channel interactions. Understanding the Protoxin-2 binding mechanism would aid in engineering a potent toxin that targets Nav1.7 without unwanted physiological effects. It is therefore our goal to map the interaction sites between Protoxin-2 and Nav1.7 VSD that are important for toxin binding. In this study, we are interested in investigating the binding of Protoxin-2 to the voltage-sensing domain of repeat 2 (VSD2) of human Nav1.7. We initially designed mutations of the VSD2 and predicted the dissociation constants of Protoxin-2 using free energy perturbations (FEP) during dynamics simulation. Microscale thermophoresis was employed to validate the FEP results by doing binding assays using the expressed Protoxin-2 and isolated VSD2 in DMPC vesicles. Monte Carlo-based molecular docking was also used to generate and screen mutations that will improve the affinity of Protoxin-2 to Nav1.7. Both the effect and affinity of selected Protoxin-2 mutants will be evaluated with whole-cell electrophysiology. Finally, we want to unravel the role of the membrane in toxin binding by studying the complex formation through solution NMR. Findings from this study would not only shed light on the molecular determinants affecting the toxin binding but also on channel gating function.