Structural insights into the folding of cohesin and the membrane binding of bactofilin

Cohesin is a highly conserved eukaryotic complex that performs two essential cellular functions: sister chromatid cohesion and DNA loop extrusion. As such, they involve the association and translocation of cohesin along chromosomes through an ATP-dependent DNA binding and release cycle. The cohesin complex is made up of Smc1, Smc3, Scc1 and additional subunits such as Scc3, Scc2, and Pds5. The Scc2 cohesin loader is involved in the “clamping” of DNA by the complex and promotes the hydrolysis of ATP through the engagement of the Smc1/3 heterodimer. Meanwhile, Scc2’s replacement by Pds5 negates the clamped state and promotes the maintenance of cohesion, thus presumably altering loop extrusion. It has recently been proposed that the putative folding of the 50 nm long stretch of the SMC coiled coils of cohesin at their elbow could play a role in these processes. I asked the questions of how cohesin folding occurs structurally and whether it is physiologically relevant. I present a series of cryo-EM structures of Saccharomyces cerevisiae cohesin that reveal the arrangement of the folded and zipped-up coils in unprecedented detail. Using these maps, I propose the precise amino acid residues coming together in the folded state, which allowed my collaborators to construct cysteine-specific crosslinks that show that cohesin folds in vivo, even during sister chromatid cohesion. In addition, my cryo-EM maps of cohesin bound to Scc2 and Pds5 show that folding enables the simultaneous interaction of those subunits with the SMC ATPase heads and hinge. The structures demonstrate that Scc2 can interact with Smc1’s ATPase head even when fully disengaged from Smc3 and that the role of K112 and K113 in the maintenance of cohesion could be explained through the binding pose of Pds5. Finally, I present a map of cohesin in its ATP engaged form that disproves that head engagement per se drives unzipping of cohesin’s coiled coil. Moreover, building upon a recent structure of β-helical bactofilin filaments, I present new insights into its filament formation and membrane binding. Bactofilins have widespread and diverse functions, from cell stalk formation in C. crescentus to chromosome segregation in M. xanthus. However, the precise molecular architecture of their filaments has remained unclear. Here, I determine the atomic coordinates of the Thermus thermophilus bactofilin (TtBac) monomer, the lack of polarity of the filament, and the strength of the β-stacking interface by designing a polymerisation-impairing mutation that enables crystallisation and structure determination. In addition, I show that the conserved N- terminal disordered tail of TtBac is responsible for direct binding to lipid membranes, both on liposomes and in E. coli cells.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2022

Author dc:creator

• Gonzalez, Andres

Advisor dc:contributor.advisor

• Löwe, Jan

• Bactofilin
• Cohesin
• Coiled coils
• Cryo-em

Licence

• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.89998

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/342580