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Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin

Nature Structural & Molecular Biology volume 18, pages 12501258 (2011) | Download Citation

Abstract

Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of α-tubulin. Tyrosination regulates recruitment of microtubule-interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP grasp-fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms.

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Acknowledgements

We thank C. Ralston for access to beamlines at the Advanced Light Source (Lawrence Berkeley Laboratories), L. Kizub for assistance with molecular biology, protein expression and purification, S. Abrams (US National Institutes of Health, NIH) for making the X. tropicalis TTL and GFP-TTL clones, early imaging efforts and the initial observation with W. Shin that TTL expression affects microtubule growth rates, W. Shin for microscopy help, R. Sunyer for early live cell imaging and running the tip tracking program setup with help from Y. Nishimura and K. Myers, and C. Waterman (NIH) for mKusabira Orange-EB3 U2OS cells. We are grateful to N. Tjandra for temporary space while our laboratory was under renovation, C. Waterman for access to microscopes and her lab's expertise, R. Levine and D.-Y. Lee for mass spectrometry analyses and access to their HPLC and S. Buchanan for crystallization incubator space. A.R.-M. thanks H. Bourne, A. Ferré-D'Amaré, S. Gottesman, E.D. Korn, N. Tjandra and C. Waterman for support and critical reading of the manuscript. The authors thank the reviewers for their helpful comments regarding the TTL effects on microtubule dynamics. A.R.-M. is a Searle Scholar and is supported by the intramural program of the National Institute of Neurological Disorders and Stroke (NINDS)/NIH.

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Affiliations

  1. Cell Biology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, USA.

    • Agnieszka Szyk
    •  & Antonina Roll-Mecak
  2. Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.

    • Alexandra M Deaconescu
  3. National Heart, Lung and Blood Institute, Bethesda, Maryland, USA.

    • Grzegorz Piszczek
    •  & Antonina Roll-Mecak

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Contributions

A.S. purified TTL and TTL mutants, obtained TTL crystals, carried out tyrosination and gel filtration assays and wrote the corresponding methods; A.M.D. processed SAXS data, obtained the reconstructions and wrote the corresponding methods; G.P. carried out and analyzed all analytical ultracentrifugation experiments and wrote the corresponding methods; A.R.-M. grew and flash-froze crystals, collected X-ray data, solved and refined the crystal structures, carried out in vitro polymerization assays and live cell imaging, and analyzed microtubule dynamics data. A.R.-M. conceived the project and planned experiments in consultation with all authors. All authors prepared figures and A.R.-M. wrote the manuscript, which was reviewed by all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Antonina Roll-Mecak.

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https://doi.org/10.1038/nsmb.2148

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