Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Redesigned proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to reengineer the multi-specific anti-apoptotic protein Bcl-xL to remove its interactions with many of its binding partners, making it a high-affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-xL sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-xL variants, we successfully identified a protein with ~ 1000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best redesigned protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems.
Submitter: Admin Admin
Submission Date: July 31, 2017, 11:46 a.m.
|Number of data points||100|
|Assays/Quantities/Protocols||Experimental Assay: Kd fBad21 ; Experimental Assay: Kd fBad23 ; Experimental Assay: Kd fBad27 ; Experimental Assay: Kd fBim27 ; Experimental Assay: Yeast display screen 1 ; Experimental Assay: Yeast display screen 2 ; Experimental Assay: Yeast display screen 3 ; Derived Quantity: SD of Kd fBad23 ; Derived Quantity: SD of Kd fBad21 ; Derived Quantity: SD of Kd fBim27 ; Derived Quantity: SD of Kd fBad27|
|Libraries||Designed library 1 ; Designed library 2 ; Kds for direct binding of Bcl-xL to peptide fBim27 (Table S4) ; Kds for direct binding of Bcl-xL to peptide fBad21 (Table S4) ; Clones from final sorted library (Table 3) ; Kds for direct binding of Bcl-xL to peptide fBad23 (Table S4) ; Kds for direct binding of Bcl-xL to peptide fBad27 (Table S4)|