title: Locating Herpesvirus Bcl-2 Homologs in the Specificity Landscape of Anti-Apoptotic Bcl-2 Proteins.
abstract: Viral homologs of the anti-apoptotic Bcl-2 proteins are highly diverged from their mammalian counterparts, yet they perform overlapping functions by binding and inhibiting BH3 (Bcl-2 homology 3)-motif-containing proteins. We investigated the BH3 binding properties of the herpesvirus Bcl-2 homologs KSBcl-2, BHRF1, and M11, as they relate to those of the human Bcl-2 homologs Mcl-1, Bfl-1, Bcl-w, Bcl-xL, and Bcl-2. Analysis of the sequence and structure of the BH3 binding grooves showed that, despite low sequence identity, M11 has structural similarities to Bcl-xL, Bcl-2, and Bcl-w. BHRF1 and KSBcl-2 are more structurally similar to Mcl-1 than to the other human proteins. Binding to human BH3-like peptides showed that KSBcl-2 has similar specificity to Mcl-1, and BHRF1 has a restricted binding profile; M11 binding preferences are distinct from those of Bcl-xL, Bcl-2, and Bcl-w. Because KSBcl-2 and BHRF1 are from human herpesviruses associated with malignancies, we screened computationally designed BH3 peptide libraries using bacterial surface display to identify selective binders of KSBcl-2 or BHRF1. The resulting peptides bound to KSBcl-2 and BHRF1 in preference to Bfl-1, Bcl-w, Bcl-xL, and Bcl-2 but showed only modest specificity over Mcl-1. Rational mutagenesis increased specificity against Mcl-1, resulting in a peptide with a dissociation constant of 2.9nM for binding to KSBcl-2 and >1000-fold specificity over other Bcl-2 proteins, as well as a peptide with >70-fold specificity for BHRF1. In addition to providing new insights into viral Bcl-2 binding specificity, this study will inform future work analyzing the interaction properties of homologous binding domains and designing specific protein interaction partners.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Glenna Wink
authors.initials: GW
authors.lastname: Foight
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: keating@mit.edu.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Keating
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D046228:Microarray Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D060066:Protein Interaction Maps; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000235:genetics; D017385:Sequence Homology; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: C083278:BHRF1 protein, Human herpesvirus 4; C474007:Bax protein (53-86); C452184:Bcl-2 protein, Human herpesvirus 8; C118862:M3 protein, Murine gammaherpesvirus; D015513:Oncogene Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014764:Viral Proteins
keywords: BH3 peptides; BHRF1; KSBcl-2; bacterial surface display; peptide design