Maps of “pocket space” sampled by individual Bcl-2 family members.

<p>The ensemble of pockets observed from simulation: individual conformations are represented as points on a two-dimensional projection that reflects the pairwise distances between their exemplars. The relative position of exemplars from experimentally-derived Bcl-xL unbound (“<i>U</i>”) and peptide-bound (“<i>P</i>”) structures are indicated, as are the positions of exemplars from Bcl-xL structures solved in complex with various inhibitors (numbers correspond to complexes listed in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.s010" target="_blank">S1 Table</a></b>). Exemplars marked “<i>D</i>” correspond to the same “distinct” conformations described in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.g005" target="_blank">Fig. 5</a></b>.</p

Comparison of “pocket space” sampled by each Bcl-2 family member.

<p>(A) A projection built using ensembles collected from simulations of several Bcl-2 family members: Bcl-xL (<i>magenta</i>), Bcl-2 (<i>green</i>), Mcl-1 (<i>red</i>), Bcl-w (<i>orange</i>), and Bax (<i>cyan</i>). Bid and Ced-9 were used in generating the projection, but for clarity are not included on this map. Target residues derived from the Bcl-xL protein interaction site were used in generating exemplars shown on the Bcl-xL and Bcl-2 MDS plots, whereas target residues derived from the Mcl-1 protein interaction site were used in generating exemplars shown on the Mcl-1 MDS plots. For each of Bcl-xL, Bcl-2, and Mcl-1 we observe a distinct region of conformational space (“<i>D</i>”) that is not sampled by any other Bcl-2 family member. (B) Comparison of an exemplar from each “distinct” region to the corresponding unbound (“<i>U</i>”) or peptide-bound (“<i>P</i>”) protein structure from which the simulation was initiated. (C) Comparison of the conformation harboring the “distinct” pocket to the corresponding unbound/peptide-bound protein structure from which the simulation was initiated.</p

Distinguishing druggable from functional sites on survivin.

<p>(<b>A</b>) The crystal structure of survivin, showing the protein interaction site (<i>red</i>) and the distal druggable site identified by NMR (<i>blue</i>) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002951#pcbi.1002951-Hajduk1" target="_blank">[14]</a>. (<b>B</b>) Volumes of surface pockets are compared for conformations generated with the biasing potential applied at random surface residues (<i>dashed black lines</i>), applied at the protein interaction site (<i>dashed red lines</i>), and applied at the distal druggable site (<i>solid blue lines</i>). Pockets emerge at the druggable site but not elsewhere on the protein surface.</p

Building “exemplars” from surface pockets.

<p>(A) Bcl-xL (<i>grey surface</i>) is shown in complex with an inhibitor (<i>spheres</i>). (B) The protein surface features a large pocket (<i>small white spheres</i>) that is complementary in shape to the inhibitor. (C) From this surface pocket, an “exemplar” is built: a map of the “perfect” ligand to complement this protein surface, without considerations of atom connectivity. The exemplar is comprised of hydrogen bond donors (<i>yellow</i>) and acceptors (<i>magenta</i>) that complement surface polar groups on the protein, and hydrophobic atoms that fill the remainder of the surface pocket (<i>cyan</i>). In the studies we report here, we will use the shape and chemical features of the exemplar as a proxy for the shape and chemical features of the protein surface pocket.</p

Opening pockets shifts the conformational ensemble towards inhibitor-bound structures.

<p>Distributions of RMSD over interface atoms (iRMSD) to the closest inhibitor-bound crystal structure for conformations generated with (<i>red lines</i>) or without (<i>black lines</i>) the biasing potential. The iRMSDs from the unbound structure to the most and least similar inhibitor-bound crystal structures are also indicated (<i>dashed brown vertical lines</i>). For all seven proteins comprising the test set, the biased simulations produced conformations closer to an inhibitor-bound crystal structure than the unbiased simulations.</p

Pocket shapes explain (+)-JQ1 selectivity across bromodomains.

<p>For each bromodomain, the exemplar distance of (+)-JQ1 to the most similar pocket is indicated by color gradient, with all distances expressed as Z-scores (<i>green</i> are most similar, <i>red</i> are most dissimilar; the range of colors for each row is normalized across that row). Markings inside the cells denote experimental binding measurements [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.ref010" target="_blank">10</a>] for each protein-ligand pair: ✔✔✔ indicates ΔTm > 7°C, ✔✔ indicates ΔTm = 3–5°C, ✔ indicates ΔTm = 0–1°C, and ✗ indicates no detectable binding. The underlying raw data are included in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.s017" target="_blank">S8 Table</a></b>.</p

Identifying surface binding pockets.

<p>(<b>A</b>) Bcl-X_L (<i>grey surface</i>) is shown in complex with an inhibitor (<i>blue sticks</i>). The protein surface features a large pocket (<i>red spheres</i>) complementary in shape to the inhibitor. (<b>B</b>) Deep pocket volumes of surface pockets at protein interaction sites harboring a bound inhibitor (<i>red line</i>) are larger than those found elsewhere on the protein surface (<i>black line</i>). Data are collected from a test set of seven proteins, each of which has been solved in complex with a small-molecule inhibitor (Bcl-X_L, IL-2, FKBP12, HPV E2, ZipA, MDM2, and the BIR3 domain of XIAP).</p

Ensembles of available pocket shapes contain distinct pocket shapes.

<p>We define the “distinctness” of a pocket as the difference in exemplar distances of the closest conformation from a different family member, and the closest conformation from one’s own ensemble. Histograms are shown over conformations that comprise the ensembles used above. By this measure, <i>all</i> known inhibitors of <i>all</i> Bcl-2 family members bind to pockets that are not unique to their cognate target protein (i.e. low “distinctness”). Data are shown for three representative Bcl-2 family members, the complete set are included as <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.s007" target="_blank">S7 Fig</a></b>.</p

Ensembles of low-energy pocket-containing conformations.

<p>(A) To examine the effect of the particular target residues used in generating the ensemble of pocket-containing conformations, we generated separate ensembles from the top-scoring pair of target residues (Ala93 and Arg139, <i>green</i>) and the second-best pair (Ala93 and Ala142, <i>red</i>). We use exemplars to compare the similarity of surface pockets on each conformation, and we show each conformation on the two-dimensional projection that best reflects the pairwise distances between them. The overlap between the two ensembles highlights the robustness of the conformations to the particular target residues. (B) For each member of the Bcl-2 family, we generated an ensemble of conformations from unbiased simulations; the distribution of these energies is shown (<i>black</i>). The range of energies for pocket-containing conformations generated using a biasing potential target residues derived from the Bcl-xL protein interaction site (<i>magenta</i>) or the Mcl-1 protein interaction site (<i>red</i>) suggest that many of these conformations are energetically accessible to these proteins under physiological conditions. All energies shown here were evaluated in the absence of the biasing potential, for fair comparison. Each simulation is started from the structure of the unbound protein; a corresponding figure starting from the peptide-bound structures containing all available complexes is available as <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.s005" target="_blank">S5 Fig</a></b>.</p

Bcl-2 family members recognize different inhibitors using distinct surface pockets.

<p>In all cases color gradient indicates the similarity between complexes, expressed as Z-scores (<i>green</i> are most similar, <i>red</i> are most dissimilar). (A) Chemical similarity of the inhibitors. (B) Three-dimensional similarity of the inhibitors’ active conformation. (C) Similarity of protein surface pockets, measured using exemplar similarity. Numbering in all cases corresponds to complexes in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.s010" target="_blank">S1 Table</a></b>. A representative subset of the complexes are included in this figure; a corresponding figure containing all available complexes is available as <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004081#pcbi.1004081.s002" target="_blank">S2 Fig</a></b>.</p