Knotting Optimization and Folding Pathways of a Go-Model with a Deep Knot

Formation of protein knots is an intriguing offshoot of the protein folding problem. Since experimental resolution on knot formation is limited, theoretical methods currently provide the most detailed insights into the knotting process. While suitable for shallow knots, molecular dynamics simulations have faced challenges capturing the formation of deep knots in proteins such as the minimally tied trefoil α/β methyltransferase from Thermotoga maritima (MTT_TM). To improve the efficiency of MTT_TM knotting in C_α Go-model simulations, mutant variants of the MTT_TM Go-model were investigated. Through a structure-based analysis of knotted and unknotted states, four residues (K71, R72, E75, V76) were identified to increase the knotting efficiency from 2% to 83% when their contact energies were doubled and dihedral strength around the knot loop increased. The key features of this model are (i) a C-terminal slipknot intermediate that threads the knot in a highly unstructured intermediate, (ii) the inability to knot in native-like intermediate states, and (iii) a minor population in a long-lived trap that cannot knot. Examination of residue 71-76 contacts provides a small set of potential mutants that can directly test the model's validity. In addition, the knotting optimization process developed here has broad applicability in generating knotting-efficient models of other knotted proteins.