Emergence of specific binding and catalysis from a designed generalist binding protein

Area of Science:

• Protein engineering and design
• Biochemistry and molecular biology
• Enzyme catalysis

Background:

• Proteins bind ligands with high specificity, but also promiscuously to other molecules.
• This promiscuity is a starting point for evolutionary adaptation and functional diversification.
• Crystallographic fragment screening is used to study protein-small molecule interactions, but not yet for de novo proteins.

Purpose of the Study:

• To investigate the binding specificity of a de novo designed protein (apixaban-binding helical bundle).
• To explore the potential for evolving new functions from weak binding interactions in designed proteins.

Main Methods:

• Application of crystallographic fragment screening to a de novo designed protein.
• Characterization of weak binding complexes.
• Engineering of distinct functions from identified binding interactions.

Main Results:

• The de novo protein formed weak complexes with small molecules, similar to natural proteins.
• These weak complexes served as starting points for designing new functions.
• Successfully engineered a turn-on fluorophore binder and a highly efficient Kemp eliminase (catalytic efficiency of 3,200,000 M⁻¹s⁻¹).

Conclusions:

• De novo designed proteins can exhibit functional evolution from weak binding interactions.
• Simultaneous exploration of sequence and chemical structure diversity is key to guiding the emergence of diverse functions in designed proteins.
• This approach offers a powerful strategy for protein design and engineering.