One-Pot N2C/C2C/N2N Ligation To Trap Weak Protein-Protein Interactions

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Weak transient protein-protein interactions (PPIs) are crucial for cellular processes but difficult to study due to their short-lived nature.
• Existing methods often struggle to capture these fleeting interactions effectively.

Purpose of the Study:

• To develop a general and facile method for unbiased trapping of weak protein-protein interactions.
• To expand the chemical ligation repertoire with novel N-terminus to N-terminus (N2N) and C-terminus to C-terminus (C2C) ligation strategies.

Main Methods:

• Proximity-induced ligations were employed to capture weak PPIs.
• Novel N2N and C2C ligation approaches were developed and combined with existing N-terminus to C-terminus (N2C) ligation.
• A one-pot strategy was utilized to link interacting proteins.

Main Results:

• The weak Ypt1:GDI interaction was successfully trapped via C2C ligation with a half-life of 4.8 minutes and near-quantitative conversion.
• The resulting Ypt1-GDI conjugate provided structural insights into GDI's lipid-binding site and Rab membrane recycling.
• The KRas homodimer, important in Ras signaling, was also trapped using this method.

Conclusions:

• The developed proximity-induced ligation strategy offers a powerful tool for studying transient PPIs.
• This method provides valuable mechanistic insights into protein interactions, such as Rab membrane recycling and KRas signaling pathways.