Interaction of the inhibitory peptides ShK and HmK with the voltage-gated potassium channel KV1.3: Role of conformational dynamics

<strong>ABSTRACT: </strong>Peptide toxins that adopt the ShK fold can inhibit the voltage-gated potassium channel K_V1.3 with IC₅₀ values in the pM range, and are therefore potential leads for drugs targeting autoimmune and neuroinflammatory diseases. NMR relaxation measurements and pressure-dependent NMR have shown that, despite being cross-linked by disulfide bonds, ShK itself is flexible in solution. This flexibility affects the local structure around the pharmacophore for K_V1.3 channel blockade and, in particular, the relative orientation of the key Lys and Tyr side chains (Lys22 and Tyr23 in ShK), and has implications for the design of K_V1.3 inhibitors. In this study, we have performed molecular dynamics (MD) simulations on ShK and a close homolog, HmK, in order to probe the conformational space occupied by the Lys and Tyr residues, and docked the different conformations with a recently determined cryo-EM structure of the K_V1.3 channel. Although ShK and HmK have 60% sequence identity, their dynamic behaviors are quite different, with ShK sampling a broad range of conformations over the course of a 5 μs MD simulation, while HmK is relatively rigid. We also investigated the importance of conformational dynamics, in particular the distance between the side chains of the key dyad Lys22 and Tyr23, for binding to K_V1.3. Although these peptides have quite different dynamics, the dyad in both adopts a similar configuration upon binding, revealing a conformational selection upon binding to K_V1.3 in the case of ShK. Intriguingly, the more flexible peptide, ShK, binds with nearly 300-fold higher affinity than HmK.