title: Stathmin and interfacial microtubule inhibitors recognize a naturally curved conformation of tubulin dimers.
abstract: Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T(2)RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T(2)RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T(2)RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T(2)RB3, T(2)Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.
authors.affiliation: Faculté de Pharmacie, Aix-Marseille Université, INSERM UMR 911, Centre de Recherche en Oncologie Biologique et Oncopharmacologie, 27 bd Jean Moulin, 13385 Marseille Cedex 05, France. pascale.barbier@univmed.fr
authors.forename: Pascale
authors.initials: P
authors.lastname: Barbier
authors.forename: Audrey
authors.initials: A
authors.lastname: Dorléans
authors.forename: Francois
authors.initials: F
authors.lastname: Devred
authors.forename: Laura
authors.initials: L
authors.lastname: Sanz
authors.forename: Diane
authors.initials: D
authors.lastname: Allegro
authors.forename: Carlos
authors.initials: C
authors.lastname: Alfonso
authors.forename: Marcel
authors.initials: M
authors.lastname: Knossow
authors.forename: Vincent
authors.initials: V
authors.lastname: Peyrot
authors.forename: Jose M
authors.initials: JM
authors.lastname: Andreu
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D008870:Microtubules; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012756:Sheep; D050777:Stathmin / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism; D014404:Tubulin / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism
chemical_list: D050777:Stathmin; D014404:Tubulin