Unique features of EIGHT fold—Proteins with the most complex 8 3 knotted topology

Herein, we present three crystal structures of the most complex knotted protein to date, an 8 3 knot with eight crossings, which belongs to a clasp knot family. The fold of this protein is also unique, and we call it the EIGHT fold (Extraordinarily Intricate Globular Helical Tangled fold). The 8 3 knot is found only in a certain group of bacteria, where it is strictly conserved, despite sequence identities as low as 10%. The 8 3 structure shows a high level of internal symmetry, with structurally analogous halves. It is possible that the EIGHT fold evolved from a single unknotted “half” that duplicated and formed a complex protein. Based on an in vitro study of three selected 8 3 proteins, we estimated their thermal and thermodynamic stability, and showed that they follow a two-state unfolding model under equilibrium conditions and their refolding occurs to a compact, nonnative state. Unfolding kinetics of 8 3 knotted proteins reveal a complex, multiphase process spanning milliseconds to days. Six kinetic phases were resolved using stopped-flow fluorescence and far-ultraviolet absorbance circular dichroism spectroscopy, highlighting both rapid early events and slower rearrangements in secondary and tertiary structures. Finally, with a structure-based model, we showed that proteins with an 8 3 knot can self-tie through a 6 1 knotted intermediate state. They can also self-untie, albeit via an additional intermediate knot such as 4 1 . Nevertheless, these proteins do not seem to fit with current folding theories known to date and require the development of a new approach.