Fig 1.
Chemical structures and tubulin binding characteristics of compounds used in this study.
(A) Amino acid constitutents of monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The four amino acids of MMAE are labeled and highlighted in color. The carboxy-terminal difference between the norephedrine group of MMAE and the phenylalanine MMAF is highlighted in pink and violetpurple, respectively. (B) Fluorescence polarization binding assay of FITC derivatives of MMAE (blue) and MMAF (red) to free tubulin. FITC-conjugated butylamine (orange) was used as negative control. Assay is conducted in 20 mM PIPES buffer pH 6.9 and 1 mM MgCl2. Ordinate values are arbitrary polarization units (P) and the abscissa denotes the log molar concentration of sheep brain tubulin. Data points are mean values from triplicate experiments with error bars representing standard deviations. KD values are 291 nM for FI-MMAE and 63 nM for FI-MMAF.
Table 1.
Data collection and refinement statistics.
Fig 2.
Location of the MMAE binding site in the T2R-TTL complex.
(A) The entire T2R-TTL complex is shown with TTL (purple) and RB3 (yelloworange), α (dark gray) and β tubulin (light gray) subunits. MMAE molecules (cyan) are bound to the complex at both the high affinity β1/α2 interface, and to the lower affinity β2 subunit alone, however density for the low affinity site is poorly resolved. (B) The high affinity MMAE binding site, colored as in panel A. The MMAE molecule makes the most extensive contacts with the H6-H7 loop on the β subunit, and the carboxy-terminal norephedrine is located directly above the GDP ligand. The amino-terminus of the molecule primarily interacts with the βT5 loop and αH10 on the adjacent subunit. (C) Specific interactions of the MMAE amino-terminal residues. Asp179 located on the T5 loop interacts with the positively charged N-methyl group and co-coordinates a crystallographic water. The side chain of Asn329 on αH10 forms a dual interaction with the amide nitrogen and carbonyl group of the MMAE valine residue. A crystallographic water molecule is also located between the MMAE valine carbonyl and the carbonyl of the Pro222 of the βH6-H7 loop. The trans-configuration observed at the Val-Dil amide bond of the bound MMAE is highlighted with a red ellipse. (D) Specific interactions of the MMAE carboxy-terminal residues. The carbonyl groups of both Dil and Dap form hydrogen bonds to the amide nitrogens of Tyr224 and Gly225, respectively. The carboxy-terminal norephedrine is positioned directly above the GDP ligand and the hydroxyl group forms interactions with the side chain of Gln15 and coordinates a crystallographic water molecule with Asn228. The SigmaA‐weighted mFo‐DFc omit map (grey mesh) in both the panels C and D is contoured at + 3.0σ.
Fig 3.
Comparison of the T2R-TTL-MMAF and the T2R-TTL-MMAE crystal structures.
(A) Superposition of the MMAE structure (white) onto the MMAF structure (dark green) highlighting the subtle differences observed at the carboxy-terminal ends in the binding site. Hydrogen bonds and waters present in the MMAF structure are represented as black dashed lines and red spheres, those of the superimposed MMAE structure are in grey. The negatively charged carboxylate of MMAF interacts with the guanidinium group of Arg278 through a crystallographic water molecule. In the MMAE structure, the carbonyl group of Dap coordinates a crystallographic water molecule located in a central position to a complex network of hydrogen bonding interactions between Arg278, Asp226 and Thr223. Both these interactions may stabilize the interaction between Arg278 and Asp226 on βH7. (B) Surface sliced view of the MMAE binding site. The surface is colored by electrostatic potential from red (-8 KbT / ec) to blue (+8 KbT / ec). The positively charged amino-terminus interacts with the relatively negative charged region of the peptide binding site. The valine and Dil sidechains orient the MMAE through interactions with hydrophobic pockets on the β1 and α2 subunits. The carboxy-terminal ends of both the MMAE and MMAF molecules are solvent exposed and the interactions with the backbone amides of the βH6-H7 loop orient their carboxy-terminal groups in a positively charged and solvent filled pocket above the bound nucleotide.
Fig 4.
The detailed tubulin-vinblastine interactions at the vinca site.
Vinblastine is in violetpurple stick representation. β1- and α2-tubulin are displayed as light and dark grey ribbons, respectively. Key residues forming the interaction with the ligand are in stick representation and are labeled. Hydrogen bonds are highlighted as dashed black lines, water molecules as red spheres.
Fig 5.
The effect of ligand binding on M-loop conformation.
Electron density map details covering the M-loops of (A) the T2R-TTL-MMAE, (B) the T2R-TTL-vinblastine and (C) the T2R-TTL-apo crystal structures. The electron density maps (blue mesh) are contoured at 1.0 σ. In both the MMAE and the vinblastine complexes, the Arg278 interaction maintains the M-loop in an extended conformation incompatible with lateral contact formation in intact microtubules. (D) Ribbon representation of the superimposed T2R-TTL-MMAE (cyan), T2R-TTL-vinblastine (violetpurple) and the T2R-TTL-zampanolide complex (orange, PDB ID 4I4T). The ligands and the highlighted residues are represented as colored sticks and are labeled. Compared to the M-loop of the T2R-TTL-zampanolide complex, which adopts a conformation that is compatible with lateral protofilament contacts, both the M-loops of the liganded MMAE and vinblastine structures adopt a more extended conformation allowing Arg278 to interact with Asp226 and through water molecules with the carboxy-terminal portions of the MMA-ligands bound to the peptide site, thereby locking the M-loop in a incompatible conformation for lateral protofilament contacts.