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

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 againstChlamydomonas 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.

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Fig: Development of monobodies against CrSAS-6: a CrSAS-6 homodimers (in blue) form ring polymers ~23 nm in diameter (left). Higher magnification views on the right show targets utilized for monobody selection: CrSAS-6_N (top) and CrSAS-6_6HR (bottom). bMonobody ribbon representation. The variable regions in the side-and-loop monobody library are colored: FG loop in red, side residues in connecting ßC/ßD strands in green and yellow, respectively, CD loop in blue. The amino acid sequences of the variable region for MBCRS6-1, MBCRS6-13 and MBCRS6-15 are shown on the right. c-e ITC profiles for the interaction between the targets CrSAS-6_6HR or CrSAS-6_N and the monobodies MBCRS6-1 (c), MBCRS6-13 (d), and MBCRS6-15 (e). f-h Structures of CrSAS-6_6HR (f) or CrSAS-6_N (g, h) in surface and ribbon representation (blue), highlighting in orange the residues interacting with MBCRS6-1 (f), MBCRS6-13 (g), and MBCRS6-15 (h), which are shown in gray in surface and ribbon representation, also in the higher magnifications on the right.

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.

Hatzopoulos, G.N., Kükenshöner, T., Banterle, N. et al. Tuning SAS-6 architecture with monobodies impairs distinct steps of centriole assembly. Nat Commun 12, 3805 (2021). https://doi.org/10.1038/s41467-021-23897-0

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