Figure 1.
Conformational rearrangements of α- and β-subunits of the straight (PDB entry 1JFF [4], resolution 3.5 Å) and bent (PDB entry 1SA0 [7], resolution 3.58 Å) tubulin heterodimers.
The N-terminal nucleotide binding (residues 1–205) and C-terminal (residues 382–437) domains are rendered as grey ribbons. Helices and β-strands comprising the intermediate domain (residues 206–381) are colored blue (straight) and red (bent). We quantify curvature of the straight and bent heterodimers based on a ∼12° intradimer rotation between the α- and β-H7 helices (residues 222–244), after superimposing the α-H7 helices. Figure was generated using PyMOL [49].
Figure 2.
Crystal structure of the colchicine-bound soluble tubulin from the bovine tubulin:RB3-stathmin-like domain (SLD) (T2R) colchicine complex.
a) The T2R-colchicine complex is a head-to-tail longitudinal assembly of two αβ-tubulin heterodimers. b) 90° rotated view of the colchicine binding site. Figure was generated using PyMOL.
Figure 3.
The (a) probability distribution, (b) free energy profile, and (c) buried surface area of unpolymerized tubulin as a function of intradimer curvature.
Intradimer rotation of the endpoint structures, the straight taxol-liganded, zinc-stabilized protofilament tubulin and bent α1β1 heterodimer from the T2R-colchicine complex, are denoted at θ = 0° (blue dash) and θ∼12° (red dash), respectively. Intradimer rotation of selected intermediately bent tubulins are denoted as dotted lines: θ∼5° (GMPCPP-tubulin in double-tube layers), θ∼6° (α2β2 heterodimer of the T2R-TTL-ADP, T2R-TTL-zampanolide, and T2R-TTL complexes), θ∼6.9° (both α1β1 and α2β2 heterodimers of the GTP-tubulin-D1 DARPin structure), and θ∼10° (α1β1 heterodimer of T2R-TTL-zampanolide and T2R-TTL epothilone A complexes). Buried surface area values of tubulin heterodimer structures with resolutions up to 3 Å are shown in panel (c); the structures from PMF in (b) (open circles) and crystal structures of the straight (solid triangle) and bent (solid circles) heterodimers are denoted for each tubulin complex.
Figure 4.
The (a) probability distribution and (b) free energy profile for the laterally-paired tubulins.
Probability histograms corresponding to each umbrella window are shown in Figure S2c in Text S1.
Table 1.
Intradimer curvature of “straight” and “bent” tubulin heterodimers.
Figure 5.
The (a) probability distribution and (b) free energy profile for the colchicine-bound tubulin as a function of intradimer rotation.
Intradimer rotation of the endpoint structures, the “straight” taxol-liganded, zinc-stabilized protofilament tubulin and “bent” α1β1 heterodimer from the T2R-colchicine complex, are denoted at θ∼0° (blue dash) and θ∼12° (red dash), respectively. Intradimer rotations observed in crystal structures of colchicine-bound tubulins are denoted as dotted lines at θ∼7.8° (both α1β1 and α2β2 heterodimers of T2R-colchicine-ustiloxin complex). Individual probability histograms corresponding to each umbrella window is shown in Figure S2b in Text S1.
Figure 6.
The colchicine binding pocket is sterically constrained in tubulin conformations with lower curvature.
a) Ratios of the volume of colchicine with respect to the volume of the predicted binding pocket for representative colchicine-bound tubulin heterodimers with curvatures of θ∼0°, 2°, 6°, 8°, 10°, and 12°. b) SiteMap predictions of the colchicine binding site for colchicine-bound tubulin structures with intradimer curvature of θ = 0°, 2°, 6°, 8°, 10°, and 12°. For each colchicine-bound tubulin structure, the representative structure with the maximum SiteScore is displayed. Black dots represent the predicted binding pocket of colchicine. Structural elements comprising the colchicine-binding domain are rendered as cartoon.