Computational redesign of protein-protein interaction specificity.

Area of Science:

• * Protein engineering and computational biology.
• * Molecular and systems biology.
• * Structural biology.

Background:

• * Protein-protein interactions (PPIs) are crucial for cellular functions.
• * Redesigning PPI specificity is challenging but essential for understanding and manipulating biological systems.
• * Existing methods for altering PPIs have limitations.

Purpose of the Study:

• * To develop and validate a 'computational second-site suppressor' strategy for redesigning PPI specificity.
• * To create novel, specifically interacting DNase-inhibitor protein pairs.
• * To demonstrate the in vitro and in vivo functionality and specificity of the designed protein interfaces.

Main Methods:

• * Computational design of protein interfaces using the second-site suppressor strategy.
• * In vitro binding and functional assays to test designed protein pairs.
• * In vivo studies in living cells to assess specificity in a natural context.
• * High-resolution X-ray crystallography to analyze the redesigned protein interface structure.

Main Results:

• * Successfully designed and generated new, specifically interacting DNase-inhibitor protein pairs.
• * Demonstrated that the designed specificity switch is effective in both in vitro and in vivo assays.
• * Obtained the first high-resolution crystal structure of a computationally redesigned, functional protein-protein interface with altered specificity.
• * Confirmed specificity of the designed interfaces within the natural functional context in living cells.

Conclusions:

• * The 'computational second-site suppressor' strategy is a viable method for redesigning protein-protein interaction specificity.
• * This approach enables the creation of novel interacting protein pairs with tailored specificities.
• * The developed method has significant potential for delineating and re-engineering protein interaction networks in living cells.