Multi-barrier unfolding of the double-knotted protein, TrmD–Tm1570, revealed by single-molecule force spectroscopy and molecular dynamics

The doubly knotted motif is one of the least expected features in proteins, occurring in both globular and transmembrane forms. Here, we focus on globular protein members of the methyltransferase family: the TrmD–Tm1570 protein, which contains two deep 3 ₁ knots, and the single-knotted proteins TrmD and Tm1570, all from Calditerrivibrio nitroreducens . Using various biophysical experimental techniques and computer simulations with AI-based methods, we studied their thermal and thermodynamic stability, as well as their mechanical unfolding. Based on molecular dynamics (MD) simulations, with the Structure-Based C α Model (SBM-C α ) and UNRES (coarse-grained), we show that native contacts alone are not sufficient to fold double-knotted proteins. However, native contacts are sufficient to fold the single-knotted proteins TrmD and Tm1570 into their native conformations. Using the same model, we identified four possible unfolding and untying pathways, in which each domain can self-tie independently at some stage of the process. Optical tweezers (OT) experiments show that this process is also reversible, although the stretched state remains knotted. In addition, we observed higher thermal and mechanical stability in Tm1570 compared with TrmD, which is partly attributable to the position of the knot core. Overall, our results suggest that double-knotted protein from the SPOUT family can only partially self-fold, and that full knotting may require the assistance of a chaperone.