Assessment of theTopology and Oligomerisation Statesof Coiled Coils Using Metadynamics with Conformational Restraints

Coiled-coil proteins provide an excellent scaffold for multistate <i>de novo</i> protein design due to their established sequence-to-structure relationships and ability to switch conformations in response to external stimuli, such as changes in pH or temperature. However, the computational design of multistate coiled-coil protein assemblies is challenging, as it requires accurate estimates of the free energy differences between multiple alternative coiled-coil conformations. Here, we demonstrate how this challenge can be tackled using metadynamics simulations with orientational, positional and conformational restraints. We show that, even for subtle sequence variations, our protocol can predict the preferred topology of coiled-coil dimers and trimers, the preferred oligomerization states of coiled-coil dimers, trimers, and tetramers, as well as the switching behavior of a pH-dependent multistate system. Our approach provides a method for predicting the stability of coiled-coil designs and offers a new framework for computing binding free energies in protein–protein and multiprotein complexes.