Binding pocket stabilization by high-throughput screening of yeast display libraries

Area of Science:

• Biochemistry
• Structural Biology
• Drug Discovery

Background:

• Protein dynamics significantly influence therapeutic target binding pockets.
• Flexible binding sites can lead to transient pocket formation, hindering drug screening.
• FK506-binding protein 51 (FKBP51) is a key target for stress-related disorders.

Purpose of the Study:

• To engineer FKBP51 variants with enhanced affinity for conformation-specific inhibitors.
• To investigate the impact of protein engineering on FKBP51 binding pocket flexibility.
• To facilitate the development of novel selective ligands for FKBP51.

Main Methods:

• Construction of yeast surface display libraries using random and site-saturation mutagenesis of the FKBP51 FK1 domain.
• High-throughput screening via fluorescence-activated cell sorting (FACS) with conformation-specific fluorescent ligands.
• Affinity determination of identified FKBP51 variants compared to wild-type.

Main Results:

• Identification of 15 FKBP51 variants with improved binding affinity to selective ligands.
• Achieved up to 34-fold increase in binding affinity compared to the wild-type protein.
• Demonstrated successful protein engineering for targeting specific ligand-bound conformations.

Conclusions:

• Engineered FKBP51 variants offer improved tools for studying binding pocket dynamics.
• These variants can aid in isolating new selective ligands targeting the open conformation of FKBP51.
• The strategy provides a pathway for developing therapeutics for stress-related disorders.