NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex

Cyprian D Cukier (1) , Audrey Tourdes (1) , Dounia El-Mazouni (1) , Valérie Guillet (1) , Julian Nomme (1) , Lionel Mourey (1) , Alain Milon (1) , Andreas Merdes (2) , Virginie Gervais (1)

1 IPBS - Institut de pharmacologie et de biologie structurale
2 CBD - Centre de biologie du développement

Mitotic-spindle organizing protein associated with a ring of c-tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of c-tubulin ring complexes (cTuRCs), which are involved in nucleation of microtubules. Despite its small size, MOZART1 represents a challenging target for detailed characterization in vitro. We described herein a protocol for efficient production of recombinant human MOZART1 in Escherichia coli and assessed the properties of the purified protein using a combination of size exclusion chromatography coupled with multiangle light scattering (SEC-MALS), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) experiments. MOZART1 forms heterogeneous oligomers in solution. We identified optimal detergent and buffer conditions for recording well resolved NMR experiments allowing nearly full protein assignment and identification of three distinct alpha-helical structured regions. Finally, using NMR, we showed that MOZART1 interacts with the N-terminus (residues 1-250) of GCP3 (c-tubulin complex protein 3). Our data illustrate the capacity of MOZART1 to form oligomers, promoting multiple contacts with a subset of protein partners in the context of microtubule nucleation.

Mots clés: GCP3; NMR; oligomeric state; c-tubulin; GCP3

Domaines: Sciences du Vivant [q-bio]

Cyprian D Cukier, Audrey Tourdes, Dounia El-Mazouni, Valérie Guillet, Julian Nomme, et al.. NMR secondary structure and interactions of recombinant human MOZART1 protein, a component of the gamma-tubulin complex. Protein Science, 2017, 26 (11), pp.2240 - 2248. ⟨10.1002/pro.3282⟩. ⟨hal-03002130⟩