title: Structure-based redesign of the binding specificity of anti-apoptotic Bcl-x(L).
abstract: 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-x(L) 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-x(L) 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-x(L) 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.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: T Scott
authors.initials: TS
authors.lastname: Chen
authors.forename: Hector
authors.initials: H
authors.lastname: Palacios
authors.forename: Amy E
authors.initials: AE
authors.lastname: Keating
mesh_terms: D000465:Algorithms; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D020650:Combinatorial Chemistry Techniques; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism*; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D013379:Substrate Specificity; D015003:Yeasts / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000378:metabolism*; D051020:bcl-X Protein / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C494819:BAD protein, human; C494811:BCL2L1 protein, human; C000606775:BCL2L11 protein, human; C474007:Bax protein (53-86); D000072224:Bcl-2-Like Protein 11; D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein