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

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.

Amino acid substitutions at proline 220 of β-tubulin confer resistance to paclitaxel and colcemid

“…Crystallographic studies indicate that the drug binds to h-tubulin near the intradimer interface where it stabilizes a ''curved'' tubulin conformation that inhibits microtubule elongation and destabilizes the polymer (13). Drugs that bind to this site are effective in treating a variety of diseases; for example, colchicine is used to reduce the inflammation associated with gout, griseofulvin is effective against some fungal infections, mebendazole is used to treat helminthiasis, and several drugs are in clinical trials for treating cancer (14,15).…”

Interactions of ataxin-3 with its molecular partners in the protein machinery that sorts protein aggregates to the aggresome

“…The oligomer is not fully straight, with the terminal dimer slightly bent relative to the axis of the preceding linear tetramer (Fig. 1A, lower panel), in a conformation reminiscent of that found in the crystal structure of tubulin in complex with the stathmin-like domain of RB3 and colchicine (PDB-code 1SA0) (Ravelli et al, 2004). The SAXS curve computed from our hexameric model yielded a good fit to the experimental data with = 1.09.…”

“…The aggregation states of the AT3Q24 and tubulin proteins were estimated from their excluded (Porod, 1982) volumes taking into account that for sufficiently large globular proteins the hydrated volume in nm 3 should numerically be about twice the molecular mass in kDa. Molecular modeling for the AT3Q24-tubulin complex was done using the NMR atomic model of the JD of AT3Q24 (PDB code; 1YZB [Nicastro et al, 2005]) and the crystallographic model of tubulin dimer (PDB code: 1TUB and 1SA0 [Nogales et al, 1998;Ravelli et al, 2004]) by manual docking to the ab initio model of the complex. The scattering pattern from the constructed model was calculated from its atomic coordinates by the program CRYSOL (Svergun et al, 1995) (see Supplementary Methods).…”

Expanding the spectrum of TUBA1A-related cortical dysgenesis to Polymicrogyria

“…However, because this mutation only adds one carbon to the aliphatic side chain, p.Val235Leu might have a more subtle effect on the piston movement of helix H7, known to control the transition of curvedstraight tubulin and thus may alter microtubule dynamics or stability. 24 According to structural data, none of these mutations seem to lie in the nucleotide-binding pocket of alpha-tubulin suggesting that they may not influence GTP binding. Additionally, they are not located at the intradimer interface, suggesting that they would not be involved in the alpha-beta heterodimerization process.…”