Tuning SAS-6 architecture with monobodies impairs distinct steps of centriole assembly

Georgios N Hatzopoulos,Niccolò Banterle,Pierre Gönczy,Tatiana Favez,Virginie Hamel,Isabelle Flückiger,Georg E Fantner,Tim Kükenshöner,Oliver Hantschel,Santiago Andany

doi:10.1038/s41467-021-23897-0

Abstract

Centrioles are evolutionarily conserved multi-protein organelles essential for forming cilia and centrosomes. Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. The importance of this architecture has been difficult to decipher notably because of the lack of precise tools to modulate the underlying assembly reaction. Here, we developed monobodies against Chlamydomonas reinhardtii SAS-6, characterizing three in detail with X-ray crystallography, atomic force microscopy and cryo-electron microscopy. This revealed distinct monobody-target interaction modes, as well as specific consequences on ring assembly and stacking. Of particular interest, monobody MBCRS6-15 induces a conformational change in CrSAS-6, resulting in the formation of a helix instead of a ring. Furthermore, we show that this alteration impairs centriole biogenesis in human cells. Overall, our findings identify monobodies as powerful molecular levers to alter the architecture of multi-protein complexes and tune centriole assembly.

Highlights

Centrioles are evolutionarily conserved multi-protein organelles essential for forming cilia and centrosomes
Centrinone acts as a binary switch for centriole assembly and is difficult to use for modulating the assembly process in a more nuanced manner
Crystal structures are available for the N-terminal globular domain of CrSAS-6, as well as for a longer polypeptide containing part of the coiled-coil domain (Fig. 1a, Supplementary Fig. 1c)

Summary

Introduction

Centrioles are evolutionarily conserved multi-protein organelles essential for forming cilia and centrosomes. Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which stack into a cartwheel that scaffolds organelle formation. The importance of this architecture has been difficult to decipher notably because of the lack of precise tools to modulate the underlying assembly reaction. SAS-6 homodimers can form higher order oligomers through a weaker interaction between two head domains from neighboring homodimers, with a ~40° angle between the two coiled-coil pairs[25,26,27] This can lead to the formation of a ninefold symmetrical ring polymer harboring 9 SAS-6 homodimers[25,26,27]. SAS-6 ring polymers possess an intrinsic ability to stack on top of one another, resulting in vertical elements whose architecture resembles that of the entire cartwheel observed in the cellular context[31]

Methods

Results

Conclusion