Volume 24, Issue 5, Pages 774-781 (May 2016) Structure-Based Design of a Novel SMYD3 Inhibitor that Bridges the SAM-and MEKK2- Binding Pockets  Glenn S. Van Aller, Alan P. Graves, Patricia A. Elkins, William G. Bonnette, Patrick J. McDevitt, Francesca Zappacosta, Roland S. Annan, Tony W. Dean, Dai-Shi Su, Christopher L. Carpenter, Helai P. Mohammad, Ryan G. Kruger  Structure  Volume 24, Issue 5, Pages 774-781 (May 2016) DOI: 10.1016/j.str.2016.03.010 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 774-781DOI: (10.1016/j.str.2016.03.010) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Processivity Kinetics for Methylation of MEKK2 Were Measured Kinetically Using LC-MS/MS (A) Spectral analysis of enzymatically digested MEKK2 at time zero (upper panel in blue), and at 5 hr post-exposure to the SMYD3 reaction (lower panel in red). The red K indicates the substrate lysine methylated by SMYD3. (B) Loss of unmethylated MEKK2 substrate was concomitant with the appearance of mono-, di-, and tri-methylated products. The relative concentration of SMYD3 used in the experiment is indicated by the dashed blue line. A co-crystal structure of SMYD3, MEKK2 peptide, and SAH. (C) MEKK2 peptide (yellow) co-crystallized in the substrate pocket of SMYD3. SMYD3 domains are color-coded as shown in the figure diagram. SAH (orange) also co-crystallized with the peptide-bound form of SMYD3. (D) A close-up view highlights key residues involved in enzyme-substrate interactions as revealed by the SMYD3 co-crystal structure with peptide and SAH bound. Hydrogen bonds are represented as dashed lines. See also Table S1 and Figure S3. Structure 2016 24, 774-781DOI: (10.1016/j.str.2016.03.010) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Structure and Inhibition Kinetics of GSK2807 (A) The structure of GSK2807 with its propyl dimethylamine side chain highlighted. (B) GSK2807 (gray) binds primarily in the SAM (S-adenosyl methionine) pocket of SMYD3. The propyl dimethylamine side chain extends into the lysine tunnel. MEKK2 peptide (yellow) is overlaid for reference. A steric clash between the propyl dimethylamine of GSK2807 and substrate lysine is predicted. Hydrogen bonds are represented as dashed lines. (C and D) Lineweaver-Burk plots of the steady-state kinetics demonstrating that GSK2807 is competitive versus SAM, and (D) noncompetitive versus full-length MEKK2 protein. Double titration experiments were perform using GSK2807 at (○) 156 nM, (●) 78 nM, (□) 39 nM, (■) 20 nM, (▵) 9.8 nM, and (▴) 0 nM at the indicated concentration of either substrate. The data suggest that a ternary complex is formed which includes MEKK2, SMYD3, and GSK2807. See also Figures S2 and S4. Structure 2016 24, 774-781DOI: (10.1016/j.str.2016.03.010) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Structural Modeling (A) A structural model of GSK2807, SMYD3, and MEKK2 peptide. Lys260 of MEKK2 is shown in two conformations: inside the lysine tunnel (yellow) and pendulated outside the lysine tunnel (orange) to accommodate the propyl dimethylamine side chain of GSK2807. (B) A close-up view highlights key interactions between the MEKK2 peptide substrate and the β hairpin loop of SMYD3. The β hairpin loop is repositioned by 4 Å in the GSK2807-SMYD3 structure (green ribbon) compared with the MEKK2-SMYD3 structure (wheat-colored ribbon). Hydrogen bonds are represented as dashed lines. Structure 2016 24, 774-781DOI: (10.1016/j.str.2016.03.010) Copyright © 2016 Elsevier Ltd Terms and Conditions