Structures of domains containing known and predicted BH3 peptides.

<p>The putative BH3 is shown in red. For Bak, Bax, Bid, POFUT2, TRPM7, PCNA, MINA, DDX4 (<i>Drosophila</i>), CASP3 and BCAR1, the structure shown is the structure of the predicted BH3-containing protein. Other BH3 motifs are highlighted in the structure of the closest CDD hit to the parent protein (domain in non-bold type). All PDB IDs are listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003693#pcbi.1003693.s007" target="_blank">Table S5</a>.</p

New tight-binding BH3 peptides.

<p>Sequence logos and multiple-sequence alignments constructed using BH3 motifs from known BH3-only/pro-apoptotic effector Bcl-2 family proteins or tight binders (K_D<500 nM) from this study. Highly conserved positions 3a and 3e are colored red. The position of the first residue of the peptide in the full-length protein follows the protein name.</p

Prediction and validation of BH3-like peptides.

<p>Proteins from the Human Protein Reference Database were scanned in 23-residue windows, sequentially aligning each window with the [abcdefg]_n heptad definition of a BH3 motif, as defined in the figure. Sequences were then filtered for amino-acid composition to give ∼600,000 candidate peptide sequences to be evaluated <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003693#pcbi.1003693-KeshavaPrasad1" target="_blank">[46]</a>. Each sequence was scored with STATIUM, STATIUM_SC, and PSSM_SPOT models for binding to each of the 5 prosurvival proteins Bcl-x_L, Bcl-w, Bcl-2, Mcl-1 and Bfl-1 (15 scores in all, for each sequence), and candidate BH3-like sequences with good scores were selected for testing on SPOT arrays, as described in the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003693#s4" target="_blank">Methods</a>. A subset of peptides with successful negative controls on the SPOT arrays was tested for binding in solution. PSSM_SPOT cartoon is for demonstration: See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003693#s4" target="_blank">Methods</a> for the references to data used to derive the model.</p

Bcl-2 receptor binding profiles of 36 BH3-like peptides from human proteins.

<p>Binding profiles for known BH3 peptides interacting with Bcl-x_L, Bcl-w, Bcl-2, Mcl-1 and Bfl-1, measured by Certo et al., are in the left panel <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003693#pcbi.1003693-Certo1" target="_blank">[10]</a>. 34 peptides identified in this study with K_D<10⁴ nM for binding to at least one of five prosurvival proteins are in the right panel; these are ordered from left to right according to binding affinity, as indicated in the greyscale key. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003693#pcbi.1003693.s005" target="_blank">Table S3</a> for the K_D values used for binning and 95% confidence intervals.</p

Predicting peptide binding to the 5 Bcl-2 receptors.

<p>The first benchmark (A) included 366 interactions (K_D<1 µM) and non-interactions (K_D>10 µM). Four models were evaluated with respect to their ability to correctly classify each example, as a function of the score cutoff used for prediction. The second benchmark (B) included 180 comparisons of one receptor binding a peptide (K_D<1 µM) and another receptor not binding that same peptide (K_D>10 µM). The difference in scores for a peptide binding to two receptors was used to predict the binding preference, and agreement with experiment was evaluated as a function of the score difference cutoff. The “PSSM_SPOT+STATIUM_SC” score is the average of the Z-scores of the two models for a given receptor. Values in parentheses report the area under the curve (AUC) for each method. For details, see the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003693#s4" target="_blank">Methods</a> section.</p