Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Aug 18;106(33):13775-9.
doi: 10.1073/pnas.0904223106. Epub 2009 Aug 5.

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

Affiliations

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

Audrey Dorléans et al. Proc Natl Acad Sci U S A. .

Abstract

Structural changes occur in the alphabeta-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a beta-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with alpha-tubulin from stacking onto a beta-tubulin beta sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the beta-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: P.M. and V.M. are employees of the company that sells Taxotere, an anti-cancer drug that targets tubulin.

Figures

Fig. 1.
Fig. 1.
Chemical formulas of colchicine and of the three colchicine domain compounds used in this study.
Fig. 2.
Fig. 2.
The conformations of the β-subunit T7 loop in soluble tubulin. (A) Overview of T2R, with the α-subunits in magenta (with bound GTP in cyan), β-subunits in green (GDP in yellow), and RB3-SLD in blue. The linker between the RB3-SLD N-terminal cap (labeled N-ter cap) and its C-terminal helix is disordered and shown as a dotted line. The colchicine-binding site, presented in B, is framed; there is an identical site at the other αβ interface. (B) The switch of the T7 loop upon colchicine binding. The β-subunit without any ligand at the colchicine site (PDB ID code: 3HKB) is in green. The T7 loop (residues 244–251) of the β-subunit with bound colchicine (yellow) is presented together with helices H7 (residues 224–243) and H8 (residues 252–260), in cyan. For clarity, only the T5 loop of the α-subunit is drawn (residues 173–182). Electron density maps of loop T7 in the unliganded structure are presented in Fig. S3.
Fig. 3.
Fig. 3.
The colchicine domain. The ligands bound to T2R are presented in cyan in their respective Fobs-Fcalc omit maps contoured either at 3 σ (A and C) or at 3.5 σ (B and D) and overlapped with colchicine (yellow). For clarity, only the T5 loop of the α-subunit is drawn (magenta). Secondary structure elements presented are defined as follows: S4 (residues 134–140), S5 (166–171), S6 (200–204), S8 (313–320), and S9 (351–356). (A) ABT751 (PDB ID code: 3HKC). (B and C) The 2 binding modes of T138067 (PDB ID code: 3HKE). The position overlapping with colchicine is presented in its electron density in B; that same position is in gray in C. A ligand covalently bound to Cys β241 is presented in its electron density in C (reversed colors). (D) TN16 (PDB ID code: 3HKD). (E) The volume occupied by colchicine domain ligands. The overlapping surfaces of colchicine and TN16 are drawn together with that of the ordered part of covalently bound T138067. They are superimposed with colchicine (yellow), TN16 (cyan), and modified Cys β241 (gray).
Fig. 4.
Fig. 4.
Structural changes in the colchicine domain upon tubulin conversion from curved to straight. (A) One β-subunit of tubulin in T2R-ABT751 is shown in pale green (N-terminal domain and C-terminal helices) and darker green (intermediate domain, from H6 to S10). ABT751 is in cyan. The intermediate domain of β-tubulin in its straight conformation (PDB ID: 1JFF) is depicted (pink) after superposition of the N-terminal domain on that of β-tubulin in T2R. In the straight structure, the intermediate domain β sheet, in particular strands S8 and S9, comes closer to the H8 helix. Together with the H7 translation, this leads to a shrinking of the colchicine domain. When a ligand is bound to it, the straight conformation cannot be accommodated owing to steric clashes with surrounding secondary structure elements (S8, S9, H7, and H8). A stereoscopic view of this Figure is presented in Fig. S4. (B) Schematic view of the conversion from curved (Left) to straight tubulin (Right). Movements of tubulin intermediate domain secondary structure elements are indicated by arrows. These movements are prevented by colchicine domain ligands (represented here as a blue disk).

Similar articles

Cited by

References

    1. Mitchison T, Kirschner M. Dynamic instability of microtubule growth. Nature. 1984;312:237–242. - PubMed
    1. Vandecandelaere A, Brune M, Webb MR, Martin SR, Bayley PM. Phosphate release during microtubule assembly: What stabilizes growing microtubules? Biochemistry. 1999;38:8179–8188. - PubMed
    1. Lowe J, Li H, Downing KH, Nogales E. Refined structure of αβ-tubulin at 3.5 A resolution. J Mol Biol. 2001;313:1045–1057. - PubMed
    1. Ravelli RBG, et al. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature. 2004;428:198–202. - PubMed
    1. Vandecandelaere A, Martin SR, Engelborghs Y. Response of microtubules to the addition of colchicine and tubulin-colchicine: Evaluation of models for the interaction of drugs with microtubules. Biochem J. 1997;323:189–196. - PMC - PubMed

Publication types

LinkOut - more resources