title: Paralog-selective ligands for bcl-2 proteins.
abstract: There is considerable current interest in molecules that bind intra- or extracellular protein surfaces and inhibit protein-protein interactions. Previously we have reported that miniature proteins based on pancreatic-fold polypeptides can recognize even shallow alpha-helix binding clefts with high affinity and selectivity against unrelated proteins. One such miniature protein, PPBH3-1, binds the anti-apoptotic protein paralogs Bcl-2 and Bcl-XL with nanomolar affinity and a DeltaDeltaG = 1.2 kcal.mol-1 preference for Bcl-XL. Here we describe the directed evolution of PPBH3-1 into two new miniature proteins, PPBH3-5 and PPBH3-6, whose paralog specificity is reversed relative to PPBH3-1. PPBH3-5 and PPBH3-6 bind Bcl-2 with nanomolar affinity and a DeltaDeltaG = 0.9-1.3 kcal.mol-1 preference for Bcl-2 over Bcl-XL. Experiments with Bcl-XL variants suggest that PPBH3-5 and PPBH3-6 achieve high paralog specificity by exploiting subtle structural or electrostatic differences in the Bcl-2 and Bcl-XL molecular landscapes. PPBH3-5 and PPBH3-6 may have unique applications as early examples of nonnatural ligands that interact selectively with Bcl-2 proteins.
authors.affiliation: Departments of Chemistry and Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.
authors.forename: Anja C
authors.initials: AC
authors.lastname: Gemperli
authors.forename: Stacey E
authors.initials: SE
authors.lastname: Rutledge
authors.forename: Abby
authors.initials: A
authors.lastname: Maranda
authors.forename: Alanna
authors.initials: A
authors.lastname: Schepartz
mesh_terms: D000595:Amino Acid Sequence; D005454:Fluorescence Polarization; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D051020:bcl-X Protein
chemical_list: D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein