Revealing Cooperation between Knotted Conformation and Dimerization in Protein Stabilization by Molecular Dynamics Simulations

The topological knot is thought to play a stabilizing role in maintaining the global fold and nature of proteins with the underlying mechanism yet to be elucidated. Given that most proteins containing trefoil knots exist and function as homodimers with a large part of the dimer interface occupied by the knotted region, we reason that the knotted conformation cooperates with dimerization in protein stabilization. Here, we take YbeA from Escherichia coli as the knotted protein model, using molecular dynamics (MD) simulations to compare the stability of two pairs of dimeric proteins having the same sequence and secondary structures but differing in the presence or absence of a trefoil knot in each subunit. The dimer interface of YbeA is identified to involve favorable contacts among three alpha-helices (alpha 1, alpha 3, and alpha 5), one of which (alpha 5) is threaded through a loop connected with a3 to form the knot. Upon removal of the knot by appropriate change of the knot-making crossing of the polypeptide chain, relevant domains are less constrained and exhibit enhanced fluctuations to decrease contacts at the interface. Unknotted subunits are less compact and undergo structural changes to ease the dimer separation. Such a stabilizing effect is evidenced by steered MD simulations, showing that the mechanical force required for dimer separation is significantly reduced by removing the knot. In addition to the knotted conformation, dimerization further improves the protein stability by restricting the alpha 1-alpha 5 separation, which is defined as a leading step for protein unfolding. These results provide important insights into the structure-function relationship of dimerization in knotted proteins.