Peptide ligands for pro-survival protein Bfl-1 from computationally guided library screening.
Pro-survival members of the Bcl-2 protein family inhibit cell death by binding short helical BH3 motifs in pro-apoptotic proteins. Mammalian pro-survival proteins Bcl-x(L), Bcl-2, Bcl-w, Mcl-1, and Bfl-1 bind with varying affinities and specificities to native BH3 motifs, engineered peptides, and small molecules. Biophysical studies have determined interaction patterns for these proteins, particularly for the most-studied family members Bcl-x(L) and Mcl-1. Bfl-1 is a pro-survival protein implicated in preventing apoptosis in leukemia, lymphoma, and melanoma. Although Bfl-1 is a promising therapeutic target, relatively little is known about its binding preferences. We explored the binding of Bfl-1 to BH3-like peptides by screening a peptide library that was designed to sample a high degree of relevant sequence diversity. Screening using yeast-surface display led to several novel high-affinity Bfl-1 binders and to thousands of putative binders identified through deep sequencing. Further screening for specificity led to identification of a peptide that bound to Bfl-1 with K(d) < 1 nM and very slow dissociation from Bfl-1 compared to other pro-survival Bcl-2 family members. A point mutation in this sequence gave a peptide with ~50 nM affinity for Bfl-1 that was selective for Bfl-1 in equilibrium binding assays. Analysis of engineered Bfl-1 binders deepens our understanding of how the binding profiles of pro-survival proteins differ and may guide the development of targeted Bfl-1 inhibitors.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Sanjib
S
Dutta
T Scott
TS
Chen
Amy E
AE
Keating
D000595:Amino Acid Sequence; D008024:Ligands; D015778:Minor Histocompatibility Antigens; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000378:metabolism*
C096816:BCL2-related protein A1; D008024:Ligands; D015778:Minor Histocompatibility Antigens; D019151:Peptide Library; D019253:Proto-Oncogene Proteins c-bcl-2