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.
Submitter: Marie Ary
Submission Date: July 6, 2017, 11:55 a.m.
|Number of data points||47751|
|Proteins||Type I cohesin domain from C. thermocellum ; Type I cohesin domain from C. cellulolyticum|
|Assays/Quantities/Protocols||Experimental Assay: Counts selected ; Experimental Assay: Interface ΔΔG (clonal titrations) ; Experimental Assay: Fitness metric ; Experimental Assay: Enrichment ratio ; Experimental Assay: Counts unselected ; Experimental Assay: Interface ΔΔG (deep sequencing) ; Derived Quantity: SD of Interface ΔΔG (clonal titrations) ; Derived Quantity: SD of Enrichment ratio ; Derived Quantity: SD of Fitness metric ; Derived Quantity: SD of Interface ΔΔG (deep sequencing)|
|Libraries||Interface ΔΔGs for Ct cohesin-Ct dockerin from yeast clonal titrations (Table S2) ; Deep mutational scan of Cc cohesin point mutants relative binding affinity to Cc dockerin (counts, enrichment ratio, fitness metric, and interface ddG (SI_TextS2&3) ; Interface ΔΔGs for Ct cohesin-Ct dockerin from yeast clonal titrations (Fig. S4a) ; Deep mutational scan of Ct cohesin point mutants on relative binding affinity to Ct dockerin (counts, enrichment ratio, fitness metric, and interface ddG (SI_TextS1)|