How knots influence properties of proteins

Molecular dynamics studies within a coarse-grained structure based model were used on two similar proteins belonging to the transcarbamylase family to probe the effects in the native structure of a knot. The first protein, N-acetylornithine transcarbamylase, contains no knot whereas human ormithine transcarbamylase contains a trefoil knot located deep within the sequence. In addition, we also analyzed a modified transferase with the knot removed by the appropriate change of a knot-making crossing of the protein chain. The studies of thermally- and mechanically-induced unfolding processes suggest a larger intrinsic stability of the protein with the knot.

💡 Research Summary

The authors investigate how a topological knot embedded in a protein influences its mechanical and thermal stability. They focus on two closely related enzymes from the transcarbamylase family: the human ornithine transcarbamylase (OTCase) with a deep trefoil (3₁) knot (PDB 1yh1) and the N‑acetylornithine transcarbamylase without a knot (PDB 1j9y). In addition, they construct a third variant, 1yh1*, by surgically removing the knot‑forming crossing in 1yh1 while preserving the overall fold. All three systems are modeled using a coarse‑grained Go‑type potential that retains native contacts and is widely employed for protein folding/unfolding simulations.

Two types of external perturbations are applied: (i) constant‑velocity pulling, mimicking atomic force microscopy (AFM) experiments, with a pulling speed of vp = 0.005 Å τ⁻¹ (≈100× faster than typical experimental rates), and (ii) constant‑force stretching. In the constant‑velocity mode the force‑extension (F‑d) curves are recorded, while in the constant‑force mode the end‑to‑end distance is monitored as a function of time. Thermal stability is probed by heating simulations that locate the folding‑temperature (Tf) where the native basin loses dominance.

Mechanical results reveal a clear advantage for the knotted protein. The peak force required to unfold 1yh1 (Fmax ≈ 3.3 ε Å⁻¹, roughly 230 pN) exceeds that for the unknotted 1j9y (Fmax ≈ 2.6 ε Å⁻¹, ~180 pN) by about 30 %. The dominant unfolding pathway for 1yh1 begins with shearing of β‑strands in domain b (G‑I, G‑L, I‑K) and is interrupted by a knot‑tightening event that creates an additional energy barrier. In contrast, 1j9y unfolds primarily by shearing strands in domain a (A‑B, A‑E). Two distinct unfolding routes are observed for each protein; a rare route for 1yh1 yields a force comparable to that of 1j9y, underscoring the stochastic nature of knot‑mediated unfolding.

Thermal simulations show that both 1yh1 and the knot‑removed 1yh1* possess higher folding temperatures than 1j9y (≈5 % increase). The presence of the knot reduces conformational entropy and adds extra native contacts, thereby stabilizing the native basin. The engineered 1yh1* retains most of the original contact map except for 14 contacts lost during the knot excision, which explains why its Tf remains slightly above that of the unknotted protein.

To contextualize the knot’s stabilizing role, the authors compare it with a disulfide bridge. By strengthening a Lennard‑Jones contact between two residues (εss = 20 ε) they mimic an effectively irreversible disulfide bond. The resulting force‑extension profile resembles that of the knotted protein, yet the disulfide cannot slide along the chain and can be chemically reduced, unlike the knot which can migrate and tighten under force. This comparison highlights that knots act as dynamic, mechanically resilient “topological cross‑links” rather than static covalent constraints.

Overall, the study provides quantitative evidence that a deep trefoil knot confers enhanced mechanical resistance and thermal robustness to a protein. The knot acts as a topological reinforcement that raises the energy barrier for unfolding, delays domain separation, and introduces characteristic knot‑tightening steps. These findings have implications for protein engineering—designing knotted proteins could improve enzyme stability under extreme conditions—and for understanding natural systems where knots may be evolutionarily selected to protect catalytic cores.