A protein engineering approach toward understanding FKBP51 conformational dynamics and mechanisms of ligand binding.

Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain. By randomizing selected regions of its ligand-binding domain and sorting yeast display libraries expressing these variants, mutants with high affinity to conformation-specific FKBP51 selective ligands were identified. These improved mutants are valuable tools for the discovery of novel selective ligands that preferentially and specifically bind the FKBP51 active site in its open conformation state. Moreover, they will help us understand the conformational dynamics and ligand binding mechanics of the FKBP51 binding pocket.

Accessing Transient Binding Pockets by Protein Engineering and Yeast Surface Display Screening.

The binding pocket of some therapeutic targets can acquire multiple conformations that, to some extent, depend on the protein dynamics and the interaction with other molecules. The inability to reach the binding pocket can impose a substantial or even insurmountable barrier for the de novo identification or optimization of small-molecule ligands. Herein, we describe a protocol for the engineering of a target protein and a yeast display FACS sorting strategy to identify protein variants with a stable transient binding pocket with improved binding for a cryptic site-specific ligand. This strategy may facilitate drug discovery using the resulting protein variants with accessible binding pockets for ligand screening.

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

Protein dynamics have a great influence on the binding pockets of some therapeutic targets. Flexible protein binding sites can result in transient binding pocket formation which might have a negative impact on drug screening efforts. Here, we describe a protein engineering strategy with FK506-binding protein 51 (FKBP51) as a model protein, which is a promising target for stress-related disorders. High-throughput screening of yeast display libraries of FKBP51 resulted in the identification of variants exhibiting higher affinity binding of conformation-specific FKBP51 selective inhibitors. The gene libraries of a random mutagenesis and site saturation mutagenesis of the FK1 domain of FKBP51 encoding sequence were used to create a yeast surface display library. Fluorescence-activated cell sorting for FKBP51 variants that bind conformation-specific fluorescently labeled ligands with high affinity allowed for the identification of 15 different protein variants with improved binding to either, or both FKBP51-specific ligands used in the screening, with improved affinities up to 34-fold compared to the wild type. These variants will pave the way to a better understanding of the conformational flexibility of the FKBP51 binding pocket and may enable the isolation of new selective ligands that preferably and selectively bind the active site of the protein in its open conformation state.