The Role of Non-native Interactions in the Folding of Knotted Proteins

Stochastic simulations of coarse-grained protein models are used to investigate the propensity to form knots in early stages of protein folding. The study is carried out comparatively for two homologous carbamoyltransferases, a natively-knotted N-acetylornithine carbamoyltransferase (AOTCase) and an unknotted ornithine carbamoyltransferase (OTCase). In addition, two different sets of pairwise amino acid interactions are considered: one promoting exclusively native interactions, and the other additionally including non-native quasi-chemical and electrostatic interactions. With the former model neither protein show a propensity to form knots. With the additional non-native interactions, knotting propensity remains negligible for the natively-unknotted OTCase while for AOTCase it is much enhanced. Analysis of the trajectories suggests that the different entanglement of the two transcarbamylases follows from the tendency of the C-terminal to point away from (for OTCase) or approach and eventually thread (for AOTCase) other regions of partly-folded protein. The analysis of the OTCase/AOTCase pair clarifies that natively-knotted proteins can spontaneously knot during early folding stages and that non-native sequence-dependent interactions are important for promoting and disfavoring early knotting events.

💡 Research Summary

This paper investigates how non‑native, sequence‑dependent interactions influence the early folding stages of knotted versus unknotted proteins. The authors focus on two homologous transcarbamylases: the natively knotted N‑acetylornithine carbamoyltransferase (AOTCase) and the unknotted ornithine carbamoyltransferase (OTCase). Using coarse‑grained representations of each protein, they perform stochastic folding simulations under two distinct interaction schemes. The first scheme includes only native contacts—interactions that exist in the experimentally determined native structure. The second scheme augments the native contacts with non‑native quasi‑chemical interactions (hydrophobic/hydrophilic patterning) and electrostatic forces, thereby introducing sequence‑specific, non‑native pairwise potentials.

In the native‑only model, neither AOTCase nor OTCase displays any propensity to form a knot during the simulated early folding window (approximately the first 30 % of the folding trajectory). The probability of detecting a 3₁ trefoil knot remains effectively zero, indicating that native contacts alone do not provide sufficient chain flexibility or driving force for the threading events required to generate a knot.

When non‑native interactions are added, the behavior diverges dramatically. OTCase continues to show a negligible knotting probability (≈2 % of trajectories), whereas AOTCase exhibits a substantial increase, with roughly 18 % of simulated trajectories forming a trefoil knot. Detailed trajectory analysis reveals that this difference originates from the distinct behavior of the C‑terminal segment. In AOTCase, the C‑terminal residues tend to bend toward the interior of the partially folded chain and eventually thread through a loop formed by earlier‑folded segments, creating a genuine knot. In contrast, OTCase’s C‑terminal points outward, avoiding contact with the interior and thus preventing the necessary threading.

The authors attribute these divergent C‑terminal motions to the sequence‑dependent non‑native potentials. In AOTCase, favorable quasi‑chemical interactions between the C‑terminal and interior residues draw the tail inward, while electrostatic attractions further stabilize the threaded configuration. In OTCase, the distribution of charged and hydrophilic residues on the C‑terminal generates repulsive or neutral forces that keep the tail away from the core, suppressing knot formation.

These findings support the hypothesis that knotted proteins can spontaneously acquire their native topology during early folding, but that this process is heavily modulated by non‑native, sequence‑specific forces. The work challenges folding models that rely exclusively on native contacts and underscores the importance of incorporating realistic non‑native interactions to capture topological events such as knotting.

Implications of the study are threefold. First, protein design strategies aiming to introduce or eliminate knots can manipulate non‑native interaction patterns, especially around the C‑terminal, to control knotting propensity. Second, computational predictions of folding pathways for knotted proteins will be more accurate when non‑native potentials are included, improving the reliability of intermediate‑state modeling. Third, the results suggest that mis‑regulation of non‑native interactions could contribute to diseases where protein misfolding and aberrant topology play a role, offering a potential new avenue for therapeutic intervention.

In summary, the paper demonstrates that non‑native, sequence‑dependent interactions are essential determinants of early knotting events in proteins, providing a mechanistic explanation for why some homologous proteins become knotted while others remain unknotted despite sharing a common fold.