Structure of the SAS-6 cartwheel hub from Leishmania major

Mark Van Breugel,Stephen H Mclaughlin,Rainer Wilcken,Christopher M Johnson,Trevor J Rutherford

doi:10.7554/elife.01812

Mark Van Breugel, Stephen H Mclaughlin + Show 3 more

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https://doi.org/10.7554/elife.01812

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Abstract

Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible. DOI: http://dx.doi.org/10.7554/eLife.01812.001.

Highlights

Eukaryotic flagella and cilia have essential roles in cell locomotion, fluid movement, and sensing
Leishmania major SAS-6 is highly similar to other SAS-6 homologues
SAS-6 organizes the rotationally ninefold symmetric cartwheel (Nakazawa et al, 2007; Kitagawa et al, 2011; van Breugel et al, 2011), an early assembly intermediate of centrioles that participates in establishing their symmetry and diameter (Nakazawa et al, 2007; Brito et al, 2012; Gonczy, 2012)

Summary

Introduction

Eukaryotic flagella and cilia have essential roles in cell locomotion, fluid movement, and sensing. Highresolution crystal structures of zebrafish and C. reinhardtii SAS-6 fragments together with biochemical and biophysical characterizations (Kitagawa et al, 2011; van Breugel et al, 2011) demonstrated that two dimerization interfaces in SAS-6 mediate this oligomerization: a coiled-coil domain that forms a rod-like parallel dimer; and a globular N-terminal domain that forms a curved head-to-head dimer Modeling these two dimer interactions together in silico resulted in ring assemblies that were compatible with the symmetry and dimension of cartwheels observed in vivo.

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