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

Cyprian D Cukier,Andreas Merdes,Audrey Tourdes,Julian Nomme,Virginie Gervais,Alain Milon,Valérie Guillet,Lionel Mourey,Dounia El‐Mazouni

doi:10.1002/pro.3282

Abstract

Mitotic‐spindle organizing protein associated with a ring of γ‐tubulin 1 (MOZART1) is an 8.5 kDa protein linked to regulation of γ‐tubulin ring complexes (γTuRCs), 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 (γ‐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.

Highlights

Microtubules are a major component of the cytoskeleton in eukaryotic cells, involved in processes such as transport, cell division, and signaling.[1]
We report the expression and purification of recombinant human MOZART1 and the structural characterization of the purified protein using a combination of dynamic light scattering (DLS), SEC-multiangle light scattering (MALS), and nuclear magnetic resonance (NMR) experiments
We describe the protocol for expression and purification of human recombinant MOZART1 protein from E. coli, which allows for obtaining sufficient amount of protein for biophysical characterization in vitro and for structural studies

Summary

Introduction

Microtubules are a major component of the cytoskeleton in eukaryotic cells, involved in processes such as transport, cell division, and signaling.[1]. The linear arrangement of tubulin dimers into protofilaments creates a polarity, with b-tubulin oriented to the so-called plus-end of the microtubule, and a-tubulin oriented toward the minus-end. Of microtubules involves hydrolysis of GTP to GDP. Polymerization of microtubules from purified a/b-tubulin is kinetically unfavorable and produces a large percentage of polymers with a variable number of protofilaments. To ensure the correct diameter and geometry, assembly of microtubules in cells is nucleated from specific sites, involving multiprotein complexes called gTuRCs (g-tubulin ring complexes).[1] Depletion of gTuRC components in cells leads to strong mitotic defects, such as aberrant mitotic spindles and errorprone segregation of chromosomes.[5]

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Results

Conclusion