Poster Presentation 23rd International Society of Magnetic Resonance Conference 2023

Structure, dynamics and stability of the smallest and most complex 71 protein knot (#237)

Shang-Te Danny Hsu 1 2 3 4 , Min-Feng Hsu 4 , Manoj Kumar Sriramoju 4 , Chih-Hsuan Lai 4 , Kai-Fa Huang 2 4 , Jing-Siou Huang 2 , Tzu-Ping Ko 4 , Yun-Ru Chen 2
  1. Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
  2. Academia Sinica Protein Clinic, Academia Sinica, Taipei, Taiwan
  3. International Institute for Sustainability with Knotted Chiral Meta Matter, Hiroshima University, Hiroshima, Japan
  4. Institute of Biological Chemsitry, Academia Sinica, Taipei, Taiwan

We showed by X-ray crystallography and solution state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 71 knotted topology that is accurately predicted by AlphaFold 2 (AF2), except for the flexible N-terminus. Such a fold has not been reported in the Protein Data Base according to a DALI search. To evaluate the accuracy of the structures of Q9PR55 determined by X-ray crystallography, NMR spectroscopy and predicted by AF2, we used ANSURR (Accuracy of NMR Structures using Random Coil Index and Rigidity) that has been used to systematically compare experimental NMR structures and the corresponding AF2 predictions. The results indicated that all three structures are equally accurate based on the experimental backbone NMR chemical shifts. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule.