Locating Herpesvirus Bcl-2 Homologs in the Specificity Landscape of Anti-Apoptotic Bcl-2 Proteins

J Mol Biol. 2015 Jul 31;427(15):2468-2490. doi: 10.1016/j.jmb.2015.05.015. Epub 2015 May 23.


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.

Keywords: BH3 peptides; BHRF1; KSBcl-2; bacterial surface display; peptide design.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Humans
  • Microarray Analysis
  • Models, Molecular
  • Molecular Sequence Data
  • Oncogene Proteins / chemistry*
  • Oncogene Proteins / genetics
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs / genetics
  • Protein Interaction Maps
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / chemistry*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Sequence Homology
  • Substrate Specificity
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics


  • BHRF1 protein, Human herpesvirus 4
  • Bax protein (53-86)
  • Bcl-2 protein, Human herpesvirus 8
  • M3 protein, Murine gammaherpesvirus
  • Oncogene Proteins
  • Peptide Fragments
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Viral Proteins