Identification of compounds that bind the centriolar protein SAS-6 and inhibit its oligomerization

Julia M.C Busch,Minos-Timotheos Matsoukas,Maria Musgaard,Georgios A Spyroulias,Philip C Biggin,Ioannis Vakonakis

doi:10.1074/jbc.ra120.014780

Julia M.C Busch, Minos-Timotheos Matsoukas + Show 4 more

Open Access

https://doi.org/10.1074/jbc.ra120.014780

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Abstract

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. Centriole assembly requires oligomerization of the essential protein spindle assembly abnormal 6 (SAS-6), which forms a structural scaffold templating the organization of further organelle components. A dimerization interaction between SAS-6 N-terminal “head” domains was previously shown to be essential for protein oligomerization in vitro and for function in centriole assembly. Here, we developed a pharmacophore model allowing us to assemble a library of low-molecular-weight ligands predicted to bind the SAS-6 head domain and inhibit protein oligomerization. We demonstrate using NMR spectroscopy that a ligand from this family binds at the head domain dimerization site of algae, nematode, and human SAS-6 variants, but also that another ligand specifically recognizes human SAS-6. Atomistic molecular dynamics simulations starting from SAS-6 head domain crystallographic structures, including that of the human head domain which we now resolve, suggest that ligand specificity derives from favorable Van der Waals interactions with a hydrophobic cavity at the dimerization site.

Highlights

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals
To identify compounds that would disrupt the spindle assembly abnormal 6 (SAS-6) head domain dimerization, we established a virtual screening process starting from the head domain dimer described for Caenorhabditis elegans SAS-6 (CeSAS-6N, residues 1– 168) [17]
We observed that addition of 2 mM A11 in analytical ultracentrifugation (AUC) assays reduced the prevalence of large protein oligomers such as tetramers derived from two CeSAS-6N-CC coiled-coil dimers interacting via their head domains (Fig. 3A)

Summary

Introduction

Centrioles are key eukaryotic organelles that are responsible for the formation of cilia and flagella, and for organizing the microtubule network and the mitotic spindle in animals. We observed that addition of 2 mM A11 in AUC assays reduced the prevalence of large protein oligomers such as tetramers derived from two CeSAS-6N-CC coiled-coil dimers interacting via their head domains (Fig. 3A).

Results

Conclusion