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

ABSTRACT: 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.

SUBMITTER: Lerma Romero JA

PROVIDER: S-EPMC9676650 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

Lerma Romero Jorge A JA Meyners Christian C Christmann Andreas A Reinbold Lisa M LM Charalampidou Anna A Hausch Felix F Kolmar Harald H

Frontiers in molecular biosciences 20221107

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 var ...[more]

PMID: 36419931

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Project description:BackgroundMetal borides are a class of inorganic solids that is much less known and investigated than for example metal oxides or intermetallics. At the same time it is a highly versatile and interesting class of compounds in terms of physical and chemical properties, like semiconductivity, ferromagnetism, or catalytic activity. This makes these substances attractive for the generation of new materials. Very little is known about the interaction between organic materials and borides. To generate nanostructured and composite materials which consist of metal borides and organic modifiers it is necessary to develop new synthetic strategies. Phage peptide display libraries are commonly used to select peptides that bind specifically to metals, metal oxides, and semiconductors. Further, these binding peptides can serve as templates to control the nucleation and growth of inorganic nanoparticles. Additionally, the combination of two different binding motifs into a single bifunctional phage could be useful for the generation of new composite materials.ResultsIn this study, we have identified a unique set of sequences that bind to amorphous and crystalline nickel boride (Ni3B) nanoparticles, from a random peptide library using the phage display technique. Using this technique, strong binders were identified that are selective for nickel boride. Sequence analysis of the peptides revealed that the sequences exhibit similar, yet subtle different patterns of amino acid usage. Although a predominant binding motif was not observed, certain charged amino acids emerged as essential in specific binding to both substrates. The 7-mer peptide sequence LGFREKE, isolated on amorphous Ni3B emerged as the best binder for both substrates. Fluorescence microscopy and atomic force microscopy confirmed the specific binding affinity of LGFREKE expressing phage to amorphous and crystalline Ni3B nanoparticles.ConclusionsThis study is, to our knowledge, the first to identify peptides that bind specifically to amorphous and to crystalline Ni3B nanoparticles. We think that the identified strong binding sequences described here could potentially serve for the utilisation of M13 phage as a viable alternative to other methods to create tailor-made boride composite materials or new catalytic surfaces by a biologically driven nano-assembly synthesis and structuring.

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

Publications

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

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