Abstract
<jats:p>The ability to redirect established protein design principles toward new supramolecular architectures remains a central challenge in peptide-based materials engineering. Here, we demonstrate that a highly designable coiled-coil motif can be reprogrammed to form cross-α filaments through inversion of the hydrophobic face of a type II heptad repeat sequence. AlphaFold-Multimer was employed to screen designed peptide to identify sequences capable of forming extended cross-α assemblies. A designed peptide, ROX-LL, self-assembled into monomorphic α-helical filaments. Cryo-electron microscopy, synchrotron SAXS, and solid-state NMR measurements revealed the presence of cross-α architecture assembled from antiparallel αhelical dimers. The resulting assembly preserved the canonical structural features of coiled-coil molecular design while adopting a fundamentally different supramolecular architecture. Individual protofilaments consisted of cross-α stacks of concatenated Alacoil-like four-helix bundles exhibiting canonical knobs-into-holes packing. These findings demonstrate that coiled-coil structural interfaces can be reprogrammed through molecular design to create a distinct filamentous protein fold and identify a novel strategy for engineering cross-α peptide assemblies.</jats:p>