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Molecular insight into γ–γ tubulin lateral interactions within the γ-tubulin ring complex (γ-TuRC)

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Abstract

γ-tubulin is essential for the nucleation and organization of mitotic microtubules in dividing cells. It is localized at the microtubule organizing centers and mitotic spindle fibres. The most well accepted hypothesis for the initiation of microtubule polymerization is that α/β-tubulin dimers add onto a γ-tubulin ring complex (γTuRC), in which adjacent γ-tubulin subunits bind to the underlying non-tubulin components of the γTuRC. This template thus determines the resulting microtubule lattice. In this study we use molecular modelling and molecular dynamics simulations, combined with computational MM-PBSA/MM-GBSA methods, to determine the extent of the lateral atomic interaction between two adjacent γ-tubulins within the γTuRC. To do this we simulated a γ–γ homodimer for 10 ns and calculated the ensemble average of binding free energies of −107.76 kcal/mol by the MM-PBSA method and of −87.12 kcal/mol by the MM-GBSA method. These highly favourable binding free energy values imply robust lateral interactions between adjacent γ-tubulin subunits in addition to their end-interactions longitudinally with other proteins of γTuRC. Although the functional reconstitution of γ-TuRC subunits and their stepwise in vitro assembly from purified components is not yet feasible, we nevertheless wanted to recognize hotspot amino acids responsible for key γ–γ interactions. Our free energy decomposition data from converting a compendium of amino acid residues identified an array of hotspot amino acids. A subset of such mutants can be expressed in vivo in living yeast. Because γTuRC is important for the growth of yeast, we could test whether this subset of the hotspot mutations support growth of yeast. Consistent with our model, γ-tubulin mutants that fall into our identified hotspot do not support yeast growth.

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Acknowledgments

The work was supported partially by funding from the United States of America National Institutes of Health R01-CA095317 (H.C. Joshi) and NRSA-GM18037 (T.W. Hendrickson). The authors are thankful to Prof. B. Jayaram and the staff of the SCFBio lab at IIT-Delhi for providing the access to their supercomputing facility to perform MD simulation. Authors are also thankful to Jaypee University of Information Technology for providing fellowship to Miss. Charu Suri.

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  1. Triscia W. Hendrickson

    Present address: Department of Biology, Morehouse College, Atlanta, GA, 30314, USA

  2. Pradeep Kumar Naik

    Present address: Department of Biotechnology, Guru Ghasidas Vishwavidyalaya (A Central University), Koni, Bilaspur, 495 009, Chhattisgarh, India

Authors and Affiliations

  1. Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Distt. Solan, 173234, Himachal Pradesh, India

    Charu Suri & Pradeep Kumar Naik

  2. Department of Cell Biology, Emory University School of Medicine, 447 Whitehead Biomedical Research Building, Atlanta, GA, 30322, USA

    Triscia W. Hendrickson & Harish C. Joshi

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  1. Charu Suri
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Suri, C., Hendrickson, T.W., Joshi, H.C. et al. Molecular insight into γ–γ tubulin lateral interactions within the γ-tubulin ring complex (γ-TuRC). J Comput Aided Mol Des 28, 961–972 (2014). https://doi.org/10.1007/s10822-014-9779-2

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