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Structural Basis for Mitotic Centrosome Assembly in Flies.

Cell (2017-06-03)
Zhe Feng, Anna Caballe, Alan Wainman, Steven Johnson, Andreas F M Haensele, Matthew A Cottee, Paul T Conduit, Susan M Lea, Jordan W Raff
RÉSUMÉ

In flies, Centrosomin (Cnn) forms a phosphorylation-dependent scaffold that recruits proteins to the mitotic centrosome, but how Cnn assembles into a scaffold is unclear. We show that scaffold assembly requires conserved leucine zipper (LZ) and Cnn-motif 2 (CM2) domains that co-assemble into a 2:2 complex in vitro. We solve the crystal structure of the LZ:CM2 complex, revealing that both proteins form helical dimers that assemble into an unusual tetramer. A slightly longer version of the LZ can form micron-scale structures with CM2, whose assembly is stimulated by Plk1 phosphorylation in vitro. Mutating individual residues that perturb LZ:CM2 tetramer assembly perturbs the formation of these micron-scale assemblies in vitro and Cnn-scaffold assembly in vivo. Thus, Cnn molecules have an intrinsic ability to form large, LZ:CM2-interaction-dependent assemblies that are critical for mitotic centrosome assembly. These studies provide the first atomic insight into a molecular interaction required for mitotic centrosome assembly.

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Adénosine 5′-triphosphate disodium salt hydrate, BioXtra, ≥99% (HPLC), from microbial
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Anti-Actin antibody, Mouse monoclonal, clone AC-40, purified from hybridoma cell culture
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B834(DE3) Competent Cells - Novagen, B834 is the parental strain for BL21. These hosts are methionine auxotrophs and allow high specific activity labeling of target proteins with 35S-methionine and selenomethionine for crystallography.