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Design and Characterization of Modular Scaffolds for Tubulin Assembly


Mignot, I; Pecqueur, L; Dorléans, A; Karuppasamy, M; Ravelli, R B G; Dreier, B; Plückthun, A; Knossow, M; Gigant, B (2012). Design and Characterization of Modular Scaffolds for Tubulin Assembly. Journal of Biological Chemistry, 287(37):31085-31094.

Abstract

In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.

Abstract

In cells, microtubule dynamics is regulated by stabilizing and destabilizing factors. Whereas proteins in both categories have been identified, their mechanism of action is rarely understood at the molecular level. This is due in part to the difficulties faced in structural approaches to obtain atomic models when tubulin is involved. Here, we design and characterize new stathmin-like domain (SLD) proteins that sequester tubulins in numbers different from two, the number of tubulins bound by stathmin or by the SLD of RB3, two stathmin family members that have been extensively studied. We established rules for the design of tight tubulin-SLD assemblies and applied them to complexes containing one to four tubulin heterodimers. Biochemical and structural experiments showed that the engineered SLDs behaved as expected. The new SLDs will be tools for structural studies of microtubule regulation. The larger complexes will be useful for cryo-electron microscopy, whereas crystallography or nuclear magnetic resonance will benefit from the 1:1 tubulin-SLD assembly. Finally, our results provide new insight into SLD function, suggesting that a major effect of these phosphorylatable proteins is the programmed release of sequestered tubulin for microtubule assembly at the specific cellular locations of members of the stathmin family.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2012
Deposited On:11 Oct 2012 14:08
Last Modified:07 Dec 2017 15:18
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Additional Information:This research was originally published in: Mignot, I; Pecqueur, L; Dorléans, A; Karuppasamy, M; Ravelli, R B G; Dreier, B; Plückthun, A; Knossow, M; Gigant, B (2012). Design and Characterization of Modular Scaffolds for Tubulin Assembly. Journal of Biological Chemistry, 287(37):31085-31094. © the American Society for Biochemistry and Molecular Biology.
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1074/jbc.M112.383869

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