Determination of binding affinity upon mutation for type I dockerin–cohesin complexes from Clostridium thermocellum and Clostridium cellulolyticum using deep sequencing

Caitlin A. Kowalsky, Timothy A. Whitehead

    Research output: Research - peer-reviewArticle

    • 1 Citations

    Abstract

    The comprehensive sequence determinants of binding affinity for type I cohesin toward dockerin from Clostridium thermocellum and Clostridium cellulolyticum was evaluated using deep mutational scanning coupled to yeast surface display. We measured the relative binding affinity to dockerin for 2970 and 2778 single point mutants of C. thermocellum and C. cellulolyticum, respectively, representing over 96% of all possible single point mutants. The interface ΔΔG for each variant was reconstructed from sequencing counts and compared with the three independent experimental methods. This reconstruction results in a narrow dynamic range of −0.8–0.5 kcal/mol. The computational software packages FoldX and Rosetta were used to predict mutations that disrupt binding by more than 0.4 kcal/mol. The area under the curve of receiver operator curves was 0.82 for FoldX and 0.77 for Rosetta, showing reasonable agreements between predictions and experimental results. Destabilizing mutations to core and rim positions were predicted with higher accuracy than support positions. This benchmark dataset may be useful for developing new computational prediction tools for the prediction of the mutational effect on binding affinities for protein–protein interactions. Experimental considerations to improve precision and range of the reconstruction method are discussed. Proteins 2016; 84:1914–1928.

    LanguageEnglish (US)
    Pages1914-1928
    Number of pages15
    JournalProteins: Structure, Function and Bioinformatics
    Volume84
    Issue number12
    DOIs
    StatePublished - Dec 1 2016

    Profile

    Clostridium cellulolyticum
    Clostridium thermocellum
    High-Throughput Nucleotide Sequencing
    Mutation
    Proteins
    cohesins
    Clostridium
    Benchmarking
    Area Under Curve
    Carrier Proteins
    Software
    Yeasts
    Datasets
    Software packages
    Yeast
    Display devices
    Scanning

    Keywords

    • cellulosomes
    • computational protein–protein interface prediction
    • deep mutational scanning
    • protein benchmark set
    • yeast surface display

    ASJC Scopus subject areas

    • Structural Biology
    • Biochemistry
    • Molecular Biology

    Cite this

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    title = "Determination of binding affinity upon mutation for type I dockerin–cohesin complexes from Clostridium thermocellum and Clostridium cellulolyticum using deep sequencing",
    abstract = "The comprehensive sequence determinants of binding affinity for type I cohesin toward dockerin from Clostridium thermocellum and Clostridium cellulolyticum was evaluated using deep mutational scanning coupled to yeast surface display. We measured the relative binding affinity to dockerin for 2970 and 2778 single point mutants of C. thermocellum and C. cellulolyticum, respectively, representing over 96% of all possible single point mutants. The interface ΔΔG for each variant was reconstructed from sequencing counts and compared with the three independent experimental methods. This reconstruction results in a narrow dynamic range of −0.8–0.5 kcal/mol. The computational software packages FoldX and Rosetta were used to predict mutations that disrupt binding by more than 0.4 kcal/mol. The area under the curve of receiver operator curves was 0.82 for FoldX and 0.77 for Rosetta, showing reasonable agreements between predictions and experimental results. Destabilizing mutations to core and rim positions were predicted with higher accuracy than support positions. This benchmark dataset may be useful for developing new computational prediction tools for the prediction of the mutational effect on binding affinities for protein–protein interactions. Experimental considerations to improve precision and range of the reconstruction method are discussed. Proteins 2016; 84:1914–1928.",
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