Slipknotting upon native-like loop formation in a trefoil knot protein

Area of Science:

• Biophysics
• Computational Biology
• Structural Biology

Background:

• Protein knots and slipknots are increasingly recognized as important structural motifs, yet their folding nucleation process remains poorly understood.
• Experimental studies indicate that de novo knot formation from extended polypeptides has not been observed, highlighting a gap in mechanistic understanding.

Purpose of the Study:

• To elucidate the complete folding mechanism of knotted proteins using theoretical and computational approaches.
• To map the free energy landscape of a knotted protein and identify key folding pathways and transition states.

Main Methods:

• Employed energy landscape theory and molecular dynamics simulations.
• Utilized an all-atom structure-based protein model to map the free energy landscape.
• Analyzed the folding mechanism through nucleation sites, free energy barriers, and distinct knot-forming routes.

Main Results:

• Identified a three-state folding mechanism characterized by a nucleation site forming a native loop and a rate-limiting barrier.
• Revealed two parallel knot-forming routes: a main slipknot pathway involving a hairpin-like C-terminal helix and a minor plug motion pathway.
• Demonstrated that knot formation is a late transition state process, with random knots being rare and unstable.

Conclusions:

• A native-biased landscape is sufficient for folding complex protein topologies.
• Proposed a generalizable folding mechanism for all known knotted protein topologies involving threading a native-like loop in a preordered intermediate.
• The findings provide critical insights into the fundamental principles governing the formation of intricate protein structures.