Journal Article Ppublish
PubMed: 42896058 DOI: 10.1073/pnas.2523017123
Unique features of EIGHT fold-Proteins with the most complex 8 3 knotted topology.
Proc Natl Acad Sci U S A 123(43):e2523017123
Authors: Mariusz Mozajew, Maciej Sikora, Mateusz Fortunka, Agata P Perlinska, Anna Kluza, Szymon Niewieczerzal, Fernando Bruno da Silva, Maciej Lukaszewicz, Julia A Sikorska, Lena J Mikulska, Sophie E Jackson, Joanna I Sulkowska
Affiliations
- Centre of New Technologies, University of Warsaw, Banacha 2C St. Warszawa 02-097, Poland.
- Centre of New Technologies, University of Warsaw, Banacha 2C St. Warszawa 02-097, Poland. Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Krakow 30-239, Poland.
- Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw 02-089, Poland.
- Centre of New Technologies, University of Warsaw, Banacha 2C St. Warszawa 02-097, Poland. Faculty of Chemistry, University of Warsaw, Warsaw 02-093, Poland.
- Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, United Kingdom.
Abstract
Herein, we present three crystal structures of the most complex knotted protein to date, an [Formula: see text] 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 [Formula: see text] knot is found only in a certain group of bacteria, where it is strictly conserved, despite sequence identities as low as 10%. The [Formula: see text] 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 [Formula: see text] 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 [Formula: see text] 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 [Formula: see text] knot can self-tie through a [Formula: see text] knotted intermediate state. They can also self-untie, albeit via an additional intermediate knot such as [Formula: see text]. Nevertheless, these proteins do not seem to fit with current folding theories known to date and require the development of a new approach.