title: Structural basis for the association of MAP6 protein with microtubules and its regulation by calmodulin.
abstract: Microtubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90-177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90-177) to microtubules with a 1:1 MAP6(90-177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90-177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca(2+)-calmodulin competes with microtubules for MAP6(90-177) binding and that the binding mode of MAP6(90-177) to microtubules and Ca(2+)-calmodulin involves a common stretch of amino acid residues on the MAP6(90-177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca(2+)-calmodulin.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM), UMR829, Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, Université Evry-Val d'Essonne, Evry 91025, France. julien.lefevre@univ-evry.fr
authors.forename: Julien
authors.initials: J
authors.lastname: Lefèvre
authors.forename: Philippe
authors.initials: P
authors.lastname: Savarin
authors.forename: Pierre
authors.initials: P
authors.lastname: Gans
authors.forename: Loïc
authors.initials: L
authors.lastname: Hamon
authors.forename: Marie-Jeanne
authors.initials: MJ
authors.lastname: Clément
authors.forename: Marie-Odile
authors.initials: MO
authors.lastname: David
authors.forename: Christophe
authors.initials: C
authors.lastname: Bosc
authors.forename: Annie
authors.initials: A
authors.lastname: Andrieux
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Curmi
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002147:Calmodulin; C491894:MAP6 protein, human; C113727:Map6 protein, rat; D008869:Microtubule-Associated Proteins; D014404:Tubulin
keywords: Calmodulin; MAP6; MAPs; Microtubules; NMR; STOP; Tubulin