Redefining the Protein Kinase Conformational Space with Machine Learning  Peter Man-Un Ung, Rayees Rahman, Avner Schlessinger  Cell Chemical Biology  Volume 25, Issue 7, Pages 916-924.e2 (July 2018) DOI: 10.1016/j.chembiol.2018.05.002 Copyright © 2018 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2018 25, 916-924. e2DOI: (10. 1016/j. chembiol Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 1 Structural Elements Determining Kinase Conformations (A) The DFG motif and αC helix adopt various conformations. (B–C) The αC-out conformation comprises a range of (B) translational and tilting movements, as well as (C) rotational movement of the αC helix. The reference αC-in conformation (PDB: 1ATP) is shown as a blue cartoon. The conserved β3-Lys/αC-Glu salt bridge interaction observed in the αC-in conformation is indicated by a yellow dashed line. Cell Chemical Biology 2018 25, 916-924.e2DOI: (10.1016/j.chembiol.2018.05.002) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 2 The Protein Kinase Conformational Space (A) Three-dimensional plot of the DFG motif vectors D1 and D2, and the αC helix vector cg_vec, of the full 3,569-structure set captures five clusters. (B) The ratios of all kinase structures in each conformation. (C–F) General features of the binding site in different conformation: (C) αC-in/DFG-in (CIDI), (D) αC-in/DFG-out (CIDO), (E) αC-out/DFG-in (CODI), and (F) αC-out/DFG-out (CODO). PDB identifiers of representative structures are in parenthesis. The αC-Glu residue, DFG-Asp residue, and the R-spine residues are represented as orange, salmon, and yellow sticks, respectively. Interactions between the conserved salt bridge residues, αC-Glu and β3-Lys (or ligand), are shown as green dashed lines. The R-spine is illustrated as a curved red dashed line. The inhibitor-binding pocket is shown as green blob. An interactive version of (A) can be found at www.schlessingerlab.org/3d-scatterplot/. Cell Chemical Biology 2018 25, 916-924.e2DOI: (10.1016/j.chembiol.2018.05.002) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 3 ωCD is an Ensemble of Conformations The primary characteristics of ωCD is the DFG-intermediate conformation, while the αC helix of the structure can adopt (A) αC-in conformation with an intact R-spine, and (B) αC-out conformation with a disrupted R-spine. The R-spine is illustrated as a curved red dashed line. Cell Chemical Biology 2018 25, 916-924.e2DOI: (10.1016/j.chembiol.2018.05.002) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 4 Distribution of Characterized Conformations Across the Kinome (A) The number indicates the number of conformations of experimentally determined structures for each kinase. The color of the circle indicates the number of unique conformations in the PDB for a particular kinase. The circle size indicates the number of structures for the particular kinase. For example, BRAF has 58 structures in five different conformations (medium-sized red circle). (B) Frequency of kinase structures in different conformations for each kinase family. The number of structures is labeled within the stacked bars; the total number of structures is listed under the family name. (C) Boxplot of the number of atomic structures for kinases versus the number of conformations for which they were determined. The number shown represents the median of the distributions. Cell Chemical Biology 2018 25, 916-924.e2DOI: (10.1016/j.chembiol.2018.05.002) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 5 Chemical Similarity Network of Kinase Inhibitors in the PDB (A) Number of unique kinase inhibitors bound to a specific kinase conformation in each kinase family. (B) Representative clusters of the kinase inhibitor similarity network generated using the Markov clustering algorithm in Cytoscape 3.5.1 (full network in Figure S7). Each node represents a small molecule in a known kinase structure, and is colored based on the conformation of the bound structure. A yellow node indicates that the inhibitor binds multiple conformations. Each edge represents the top 5% values among all pairwise comparisons. Pairwise similarity of average Tanimoto coefficient of 0.47 of three chemical fingerprints was used as cutoff to define an edge. Relevant clusters are marked with black circles and italic roman numbers. (C) Representative scaffolds and chemical structures are shown for selected clusters. The PDB code of the compound (e.g., FLW), the common name (in parentheses), as well as the inhibitor type are listed. Cell Chemical Biology 2018 25, 916-924.e2DOI: (10.1016/j.chembiol.2018.05.002) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 6 Conformation-Specific Chemical Substructures (A) Enrichment of the conformation-specific chemical substructures derived from the inhibitor-bound kinase structures. The red line represents the standard p value cutoff at 0.05, and the purple line represents the Bonferroni corrected p value cutoff at 4.7 × 10−6. (B) Examples of the enriched inhibitor substructures found in each conformation. As a special case, a substructure associated with type-III kinase inhibitors is listed separately from CODI-specific substructures. Ranking of the substructures in the enrichment is listed in parenthesis. p is the uncorrected significant p value. An interactive version of this figure can be found at www.schlessingerlab.org/substructure. Cell Chemical Biology 2018 25, 916-924.e2DOI: (10.1016/j.chembiol.2018.05.002) Copyright © 2018 Elsevier Ltd Terms and Conditions