摘要
YbeA from E. coli is a trefoil-knotted SpoU-TrmD (SPOUT) RNA methyltransferase. While its knotted motif plays a key functional role, it is unclear how the knotted topology emerged from evolution. Here, we reverse-engineered an unknotted circular permutant (CP) of YbeA by introducing a new opening at the knotting loop. The resulting CP folded into an unexpected domain-swapped dimer. Untying the knotted loop abrogated its function, perturbed its folding stability and kinetics, and induced allosteric dynamic changes. We speculated that the knotted loop of YbeA is under tension to keep the cofactor in a high-energy configuration while keeping the threading C-terminal helix being knotted. Circular permutation released the mechanical strain thereby allowing the spring-loaded threading helix to flip, to relax, and to form a domain-swapped dimer. Being knotted may be the consequence of selection pressure for the unique structure-function relationship of the SPOUT superfamily that exists in all kingdoms of life. An unknotted circular permutant of YbeA was generated to understand how the conserved knotted topology emerged from evolution. Induction of a circular permutation at the knotting loop relieved the mechanical strain essential for maintaining the cofactor in a high-energy configuration, and resulted in a domain-swapped dimer.