Structural Basis of cis- and trans-Combretastatin Binding to Tubulin Roberto Gaspari, Andrea E. Prota, Katja Bargsten, Andrea Cavalli, Michel O. Steinmetz  Chem  Volume 2, Issue 1, Pages 102-113 (January 2017) DOI: 10.1016/j.chempr.2016.12.005 Copyright © 2017 Elsevier Inc. Terms and Conditions

Chem 2017 2, 102-113DOI: (10.1016/j.chempr.2016.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Chemical Structures of Ligands and Overall View of the Structure of the T2R-TTL-cis-CA-4 Complex (A) Chemical structures of combretastatin A4 (left) and colchicine (right). (B) Ribbon representations of α- and β-tubulin (dark and light gray, respectively), TTL (blue), and RB3 (brown). The tubulin-bound cis-CA-4 molecules are represented as yellow spheres. The electron-density maps highlighting the bound cis-CA-4 are displayed in the dotted box. The SigmaA-weighted 2mFo − DFc (dark blue mesh) and mFo − DFc (light green mesh) omit maps are contoured at +1.0σ and +3.0σ, respectively. The map calculations excluded the atoms of the cis-CA-4 ligand. See also Table S1. Chem 2017 2, 102-113DOI: (10.1016/j.chempr.2016.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Tubulin-cis-CA-4 Interaction Mode as Revealed by X-Ray Crystallography (A) Close-up view of cis-CA-4 (yellow) bound to the colchicine site of tubulin (gray ribbon; PDB: 5LYJ). (B) Side view of the same superposition as in (A). Note that residues βVal238 and βIle378, which are mentioned in the main text, are positioned behind the β strand βS8 and are thus not visible. (C) Superimposition of the tubulin-cis-CA-4 (yellow and gray) and tubulin-colchicine (light blue; PDB: 4O2B) complex structures in the same orientation and representation as in (A). (D) Side view of the same superposition as in (C). For simplicity, only α-tubulin residues and secondary structure elements are indicated with an α. See also Figures S1 and S3. Chem 2017 2, 102-113DOI: (10.1016/j.chempr.2016.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Metadynamic Simulations of the cis and trans CA-4 Isomers Bound to Tubulin (A) Description of CV1 and CV2 in terms of torsional angles of the ligand. (B) Free-energy surface (color map) as a function of the collective variables CV1 and CV2, described in (A). The position of the cis-CA-4 conformation in the free-energy map as seen in the tubulin-cis-CA-4 crystal structure is indicated by a purple diamond. (C) Ligand conformations corresponding to the free-energy minima C1, C2, T1, T2, and T3. For simplicity, only α-tubulin residues are indicated with an α. See also Figures S2 and S5. Chem 2017 2, 102-113DOI: (10.1016/j.chempr.2016.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Simulated Thermodynamic Cycle Illustration of the thermodynamic cycle used for computing the relative binding free energy: ΔΔGbindingtrans-cis=ΔGbindingtrans−ΔGbindingcis=ΔGunboundtrans-cis−ΔGboundtrans-cis. CV1 represents the torsional angle around the carbon-carbon double bond of CA-4 and spans from 0 (cis) to ±π (trans). The error bar is defined as the energy range spanned by three independent metadynamics runs over the last 25 ns of the simulation. See also Figures S6 and S7. Chem 2017 2, 102-113DOI: (10.1016/j.chempr.2016.12.005) Copyright © 2017 Elsevier Inc. Terms and Conditions