PDBsum entry 1sa0

Cell cycle

PDB id: 1sa0

Contents

Protein chains

427 a.a. *

419 a.a. *

124 a.a. *

Ligands

GTP ×2

GDP ×2

CN2 ×2

Metals

_MG ×3

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Insight into tubulin regulation from a complex with colchicine and a stathmin-Like domain.

Authors

R.B.Ravelli, B.Gigant, P.A.Curmi, I.Jourdain, S.Lachkar, A.Sobel, M.Knossow.

Ref.

Nature, 2004, 428, 198-202. [DOI no: 10.1038/nature02393]

PubMed id

15014504

Abstract

Microtubules are cytoskeletal polymers of tubulin involved in many cellular functions. Their dynamic instability is controlled by numerous compounds and proteins, including colchicine and stathmin family proteins. The way in which microtubule instability is regulated at the molecular level has remained elusive, mainly because of the lack of appropriate structural data. Here, we present the structure, at 3.5 A resolution, of tubulin in complex with colchicine and with the stathmin-like domain (SLD) of RB3. It shows the interaction of RB3-SLD with two tubulin heterodimers in a curved complex capped by the SLD amino-terminal domain, which prevents the incorporation of the complexed tubulin into microtubules. A comparison with the structure of tubulin in protofilaments shows changes in the subunits of tubulin as it switches from its straight conformation to a curved one. These changes correlate with the loss of lateral contacts and provide a rationale for the rapid microtubule depolymerization characteristic of dynamic instability. Moreover, the tubulin-colchicine complex sheds light on the mechanism of colchicine's activity: we show that colchicine binds at a location where it prevents curved tubulin from adopting a straight structure, which inhibits assembly.

Figure 1.
Figure 1: The tubulin-colchicine:RB3-SLD complex. a, The complex includes two tubulin heterodimers, with colchicine bound to subunits at the interface with . The RB3-SLD connecting region (residues 29-45) is from tubulin-podophyllotoxin:RB3-SLD, where it is clearest (podophyllotoxin is a competitive inhibitor of colchicine binding to tubulin23). b, The hairpin (orange) in the N-terminal domain of RB3-SLD caps the T2R complex, extending the sheet (yellow) of the intermediate domain in the 1 subunit. The extensive overlap with a protofilament (+ )-end subunit15, preventing the addition of the T2R complex to a microtubule, is illustrated. c, Interactions of RB3-SLD residues with tubulin (except for the least-well-defined RB3-SLD connecting region and for the extension of the tubulin intermediate domain -sheet) represented by yellow connecting areas. Red bars, residues of the -helix pointing towards tubulin. Dashed lines, main-chain hydrogen bonds in the extension of the intermediate domain -sheet. Within the internal repeat (grey), identical residues are connected in blue (thick blue, side-chain pointing towards tubulin). Asterisks indicate positions of stathmin phosphorylation sites.

Figure 3.
Figure 3: The colchicine-binding site on tubulin. a, DAMA-colchicine superimposed on electron-density maps. The 3.5 Å F[obs]-F[calc] omit map (cyan) is contoured at 3 . The 4 Å F[obs]-F[calc] maps calculated with colchicine (red, contoured at 3 ) and with DAMA-colchicine but F[obs] of a tubulin-colchicine:RB3-SLD complex (green, contoured at -3.3 ) are also presented. b, The colchicine site in the tubulin-colchicine:RB3-SLD complex (bright colours: tubulin loops and secondary structure elements contacting colchicine). c, Interference between colchicine binding and the straight conformation of tubulin in protofilaments. An subunit is positioned near a colchicine-bound subunit as across an intradimer longitudinal contact in a protofilament. The -tubulin subunit is prevented from occupying this position because of: (1) steric hindrance between colchicine and residues 101, 181 and GTP; and (2) colchicine forcing the T7 loop, H8 helix (for clarity, only side chains of two interfering residues-- 71 and 251--are presented) and the Lys 352 side chain to interfere with -tubulin.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2004, 428, 198-202) copyright 2004.