Loop Modulates Long-Range Allosteric Communications in a Knotted Protein

Knots in proteins are interesting topological motifs whose biological objective is not fully understood: whether they impart structural stability, refine catalysis, or control dynamics. Here, we investigate the role of a trefoil knot in N-acetyl-l-ornithine transcarbamoylase (AOTCase), especially the knotted loop and pivotal residue N183, in regulating long-range allosteric communication with the active site. Using classical molecular dynamics simulations and network-based correlation analysis, we observed that a redundant set of communication routes is followed in the ligand-bound state, passing through the knotted loop, and the corresponding thread residues are absent in the apo state. Mutating residue 183 to alanine (N183A) collapses this network, redistributing the major communication hubs away from the knotted loop. Loss in allostery is also highlighted by the pathways becoming shorter and fewer. Free energy surfaces, along with principal component analysis, reveal higher conformational restrictions in the mutated proteins. Simultaneously, contact-based analysis shows that the knotted loop and thread gap are tightened. Our work demonstrates that along with providing structural shielding, the knotted loop can also modulate allosteric networks, where N183 acts as the tunable node, opening new avenues in understanding the utility of knots in proteins.