Elucidating Molecular Mechanisms of Protoxin-2 State-specific Binding to the Human NaV1.7 Channel

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula protoxin-2 (PTx2) has high selectivity for hNaV1.7 and serves as a valuable scaffold to design novel therapeutics to treat pain. Here, we used computational modeling methods to study the molecular mechanisms by which PTx2 exhibits state-dependent binding to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic structural models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to VSD II and IV in the deactivated conformations. These state-specific interactions are mediated primarily by PTx2 R22, K26, and K27 residues with the VSD S3-S4 loop, and L23, W24 with the S1-S2 loop. The surrounding membrane lipids facilitated the binding process. Our work revealed important protein-protein contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.SummaryVoltage-gated sodium channel NaV1.7 plays a major role in pain perception and is selectively inhibited by tarantula venom protoxin-2, used to design pain therapeutics. Here, Ngoet alapplied computational modeling to assess state-dependent protoxin-2 - NaV1.7 binding.