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

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

Glenna Wink Foight  1 , Amy E Keating  2
Affiliations

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

Glenna Wink Foight et al. J Mol Biol. .

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.

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

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Figures

Figure 1
Figure 1
Sequence comparison of 5 human and 3 viral Bcl-2 homologs. (a) Percent sequence identity over the entire Bcl-2 domain (without the C-terminal trans-membrane helices or the N-terminal PEST domain of Mcl-1). (b) Percent sequence identity for residues in the BH3 binding groove (see Materials and Methods). (c) Sequence alignment used for the sequence identity calculations. Dots under residues denote residues in the BH3 binding groove. Motifs discussed in the Results section are colored and/or underlined.
Figure 2
Figure 2
Comparison of the physicochemical characteristics of human and viral Bcl-2 homolog BH3 binding groove structures using SiteMAP. The intersection score is a measure of similarity that takes into account the hydrophobic and hydrogen-bond donor/acceptor characteristics of regions of the binding site (see Materials and Methods for details of the metric). The structures of eight Bcl-2 homologs were clustered according to the similarity of their intersection score profiles. Boxed regions partition receptors or groups of receptors; numbers are the average log10(intersection score) over the indicated box and its symmetry-related box across the diagonal. PDB IDs are given, with the receptor name next to them. All mammalian receptors are the human homologs except for several murine Bfl-1 structures (“mBfl-1”). The suffix “min” denotes a structure relaxed without the peptide bound. Asterisks denote homology models.
Figure 3
Figure 3
Comparison of BH3 peptide binding profiles for eight Bcl-2 homologs. (a) Interactions with functionally validated BH3 peptides, using data from the literature. (b) Interactions with 36 BH3-like peptides identified from the human proteome. Receptors are clustered based on correlation of their binding profiles, and peptides are clustered by Euclidean distance. Dissociation constants for the human proteins in panel B were taken from DeBartolo et al. Asterisks denote peptide clusters with tighter binding to KSBcl-2 than to Mcl-1. The heat map indicates affinity measured by fluorescence anisotropy as log10 (KD in nM), with white indicating no detectable binding up to 3000 nM. KD values with 95% confidence intervals are given in Supplementary Table 1 for the 36 BH3-like peptides.
Figure 4
Figure 4
Bim BH3 and point-mutant peptides on SPOT arrays binding to 100 nM BHRF1 or KSBcl-2. Each row is labeled with the wild-type Bim residue and the position that was varied, and columns are labeled with the substitution. The column labeled “X” included peptides with the following BH3 sequences, from top to bottom: Bad, Bid, Bmf, Hrk, Noxa, Bik, Bak, Bax, Bcl-xL, Bcl-w, Bcl-2, Mcl-1, Bfl-1, BHRF1, KSBcl-2.
Figure 5
Figure 5
Bacterial surface display screen for selective binders of KSBcl-2 and BHRF1. (a) The libraries were displayed on the N-terminus of eCPX. A FLAG tag was displayed N-terminal to the peptide, and peptide expression was detected by an anti-FLAG antibody conjugated to APC. Streptavidin-phycoerythrin (SAPE) was used to detect binding of biotinylated Bcl-2 proteins. (b) Representative FACS plot of wild-type Bim BH3 displayed on the surface of E. coli binding to 5 nM biotinylated KSBcl-2. Binding, as reported by PE fluorescence, is plotted as a function of expression reported by APC fluorescence. The lower-left quadrant includes non-expressing cells. (c) equence logo built from deep sequencing of the naïve and eighth (final) library pools, using unique sequences. Heptad positions are indicated, and the wild-type Bim residue is given at each position below the logo. Pool 8 sequences included in these logos were filtered to include only those sequences also present in pools 6 and 7.
Figure 6
Figure 6
Enrichment of residues in library sequences versus viral Bcl-2 and Mcl-1 model scores. Each point represents a residue and is colored according to enrichment (log2 (frequency in final pool/frequency in naïve pool)). The frequencies were calculated from unique sequences filtered as described in the Materials and Methods. Residues in the KSBcl-2 library (a, b) and BHRF1 library (c, d) are plotted by (a, c) PSSMSPOT scores and (b, d) ΔSTATIUM scores. For all models, a score of zero is given for the wild-type residue and scores greater than zero indicate tighter binding than wild type.
Figure 7
Figure 7
Binding of human and viral Bcl-2 homologs to peptides identified from library screening. KD values, measured by fluorescence anisotropy, here plotted as log10 (KD in nM), are given with 95% confidence intervals in Supplementary Table 6. White indicates no binding up to 1000 nM. Library peptide sequences are shown to the right, with the heptad register indicated above and with varied positions underlined. Bcl-2 protein receptors (in columns) were clustered by the correlation of their binding profiles and peptides (in rows) were clustered by Euclidean distance.
Figure 8
Figure 8
Specificity mechanisms employed by library peptides. (a) PSSMSPOT scores for substitutions at Bim peptide positions L3a, I3d, and F4a. (b) SiteMAP hydrophobic density near position 3a shows extra density (circled) for KSBcl-2, BHRF1, and Mcl-1 (red, green, and blue, respectively). Bim with leucine at position 3a (2PQK) is in gray, and isoleucine at 3a from an Mcl-1-specific peptide (3KZ0) is shown in blue. The threonine on helix 5 and valine on helix 4 from 3KZ0 are also shown in blue. (c) SiteMAP similarity score for the region around position 3d, clustered by structure. Black lines divide clusters, and Mcl-1 structures form a cluster distinct from the other receptors. (d) SiteMAPs for BHRF1 and Bfl-1 have significant donor density near the peptide 3g position.
Figure 9
Figure 9
Specificity mechanisms that disfavor Mcl-1 binding. (a) Natural BH3-like sequences showing specificity for binding KSBcl-2 over Mcl-1 have diverse residues at position 4e (highlighted), including positively charged residues. (b) Tyrosine at position 4e in an Mcl-1:Bim BH3 complex (2PQK, blue:gray). (c) Aspartate at position 3f in the same Mcl-1:Bim BH3 complex.
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