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Journal Articles Journal of Biological Chemistry Year : 2013

Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin

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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.
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Dates and versions

inserm-00858655 , version 1 (07-07-2014)

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Julien Lefèvre, Philippe Savarin, Pierre Gans, Loïc Hamon, Marie-Jeanne Clément, et al.. Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin: Microtubule and calmodulin binding on Mn modules of MAP6. Journal of Biological Chemistry, 2013, 288 (34), pp.24910-24922. ⟨10.1074/jbc.M113.457267⟩. ⟨inserm-00858655⟩
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