The Role of Non-Native Interactions in the Folding of Knotted Proteins

Stochastic simulations of coarse-grained protein models are used to investigate the propensity to form knots in early stages of protein folding. The study is carried out comparatively for two homologous carbamoyltransferases, a natively-knotted N-acetylornithine carbamoyltransferase (AOTCase) and an unknotted ornithine carbamoyltransferase (OTCase). In addition, two different sets of pairwise amino acid interactions are considered: one promoting exclusively native interactions, and the other additionally including non-native quasi-chemical and electrostatic interactions. With the former model neither protein shows a propensity to form knots. With the additional non-native interactions, knotting propensity remains negligible for the natively-unknotted OTCase while for AOTCase it is much enhanced. Analysis of the trajectories suggests that the different entanglement of the two transcarbamylases follows from the tendency of the C-terminal to point away from (for OTCase) or approach and eventually thread (for AOTCase) other regions of partly-folded protein. The analysis of the OTCase/AOTCase pair clarifies that natively-knotted proteins can spontaneously knot during early folding stages and that non-native sequence-dependent interactions are important for promoting and disfavouring early knotting events. Author Summary: Knotted proteins provide an ideal ground for examining how amino acid interactions (which are local) can favor their folding into a native state of non-trivial topology (which is a global property). Some of the mechanisms that can aid knot formation are investigated here by comparing coarse-grained folding simulations of two enzymes that are structurally similar, and yet have natively knotted and unknotted states, respectively. In folding simulations that exclusively promote the formation of native contacts, neither protein forms knots. Strikingly, when sequence-dependent non-native interactions between amino acids are introduced, one observes knotting events but only for the natively-knotted protein. The results support the importance of non-native interactions in favoring or disfavoring knotting events in the early stages of folding.