The Gordian Knot Enhances Ubiquitin Binding in UCH-L1

UCH-L1 is a monomeric deubiquitinating enzyme whose native structure embeds a shallow 52 knot near the N-terminus, placing the knotted topology in direct proximity to both the substrate-binding pocket and the catalytic site. While our previous work ( J. Chem. Inf. Model.2024, 64, 6827−6837.) established that N-terminal integrity is critical for catalytic activity, the energetic and structural consequences of removing the knot without altering the amino acid sequence remained unresolved. Here, we combine steered molecular dynamics with umbrella sampling to generate full-length, sequence-identical variants of UCH-L1 with weakened or disrupted topology and reconstruct the free-energy profile along a constrained unthreading pathway. The potential of mean force places the sampled unknotted conformations approximately 5–7 kcal/mol above the native knotted minimum, indicating a thermodynamic preference for the native topology along this pathway. Long unbiased MD simulations of fully unknotted variants in both apo and holo states show that knot removal predominantly increases local N-terminal flexibility without significantly destabilizing the global fold or disrupting the catalytic-site geometry. MM-PBSA calculations further predict less favorable ubiquitin-binding energetics upon unknotting (ca. −62 vs ca. −76 kcal/mol), suggesting that topology contributes to substrate affinity. Together, these results suggest that the 52 knot in UCH-L1 is not a passive topological feature but a functional element that constrains unfolding dynamics and contributes to substrate binding efficiency.