Determinants of protein-peptide interaction specificity in the Bcl-2 and TRAF families

Abstract

Protein-peptide interactions have important roles in the majority of cellular processes. There are many families of peptide recognition domains in which homologous members display differential binding preferences for peptide sequence features. Peptide binding specificity is critical for the functional roles played by each family member, which can be overlapping or distinct. The two peptide recognition domain families discussed in this work, Bcl-2 and TRAF proteins, have roles in cellular processes including apoptosis, inflammation, and immunity. Aberrant function of these proteins has been linked to a variety of diseases. There is great interest in understanding the mechanistic basis of protein-peptide binding specificity in these families and others. An improved understanding will enable models of binding preferences for interactome prediction and design of specific peptide reagents for the inhibition and study of protein-peptide interactions. The anti-apoptotic Bcl-2 family members bind [alpha]-helical Bcl-2-homology 3 (BH3) motifs in pro-apoptotic Bcl-2 family members to prevent apoptosis. Kaposi Sarcoma herpesvirus and Epstein Barr herpesvirus express viral homologs of the anti-apoptotic Bcl-2 proteins, KSBcl-2 and BHRF 1, respectively, during viral replication to prevent host cell death. Because human Bel- 2 proteins are important in preventing apoptosis in cancers, there is interest in targeting the viral homologs, as they may also have a role in herpesvirus-associated malignancies. I designed and screened libraries of BH3 peptide variants for binding specificity to KSBcl-2 and BHRF 1. From library screening and additional rational mutagenesis, I developed peptides that showed specific binding to KSBcl-2, BHRF 1, or the human homolog Ml- 1, and displayed large margins of specificity over the other human Bel-2 homologs. TRAF proteins bind sequences in the unstructured regions of cell surface receptors and other adapter proteins in order to mediate downstream signaling events. TRAF-peptide binding preferences are relatively uncharacterized. I adapted a bacterial surface display system for screening peptides for TRAF binding. Using this system, I explored the binding preferences of TRAFs 2, 3, and 5 to single and double mutant libraries of two peptide interaction partners from CD40 and TANK. Comparison of the enriched peptide sequences reveals a surprising degree of difference between these three close TRAF homologs, yielding hypotheses relevant to TRAF function and inhibition.