What AlphaFold tells us about cohesin’s retention on and release from chromosomes

Reviewer #1 (Public Review):

There are a number of outstanding questions concerning how cohesin turnover on DNA is controlled by various accessory factors and how such turnover is controlled by post-translational modification. In this paper, Nasmyth et al. perform a series of AlphaFold structure predictions that aim to address several of these outstanding questions. Their structure predictions suggest that the release factor WAPL forms a ternary complex with PDS5 and SA/SCC3. This ternary complex appears to be able to bind the N-terminal end of SCC1, suggesting how formation of such a complex could stabilize an open state of the cohesin ring. Additional calculations suggest how the Eco/ESCO acetyltransferases and Sororin engage the SMC3 head domain and thereby protect against WAPL-mediated release.

This work thus demonstrates the power of AF prediction methods and how they can lead to a number of interesting and testable hypotheses that can transform our understanding of cohesin regulation. These findings require orthogonal experimental validation, but the authors argue convincingly that such validation should not be a pre-requisite to publication.

As the authors did not systematically include model confidence scores it is difficult for the reader to evaluate the reliability of the models obtained. The caveat is that many readers will by default assume that the presented models are correct, when in fact, some of them may score poorly and require careful assessment. As numerous readers will not be very familiar with the AF confidence scoring mechanisms, it would be important to include such metrics and indicate what these scores mean for the different interfaces (Acceptable, Medium and High confidence?). pLDDT and PAE plots should be included. When they report on a key interaction (E.g. WAPL-SCC1) they should indicate the key region (SCC1 N-terminus) on the PAE plot. False positives are always possible even with good scores, especially when many protein pairs are tried. It would therefore be important to also include a table showing the global scores for pTM and ipTM to summarise the confidence scores of interfaces.

It is exciting to see AF-multimer predictions being applied to cohesin. As some of the reported interactions are not universally conserved and some involve relatively small interfaces the possibility arises that these interfaces show poor or borderline confidence scores. As some of these interfaces map to mutants that have previously been obtained by hypothesis-free genetic screens and mutational analyses they appear nevertheless valid. Thus, an important point to make is that even interfaces that show modest confidence scores may turn out to be valid while others may be not. The authors therefore should emphasize that the proposed models are just predictions and that additional orthogonal validations are required.

Reviewer #2 (Public Review):

The ATPase protein machine cohesin shapes the genome by loop extrusion and holds sister chromatids together by topological entrapment. When executing these functions, cohesin is tightly regulated by multiple cofactors, such as Scc2/Nipbl, Pds5, Wapl, and Eco1/Esco1/2, and it undergoes dynamic conformational changes with ATP binding and hydrolysis. The mechanisms by which cohesin extrudes DNA loops and medicates siter-chromatid cohesion are still not understood. A major reason for the lack of understanding of cohesin dynamics and regulation is the failure to capture the structures of intact cohesin in different nucleotide-bound states and in complex with various regulators. So far only the ATP state cohesin bound to NIPBL and DNA have been experimentally determined.

In this manuscript, Nasmyth et al. made use of the powerful protein structure prediction tool, AlphaFold2 (AF), to predict the models of tens of cohesin subcomplexes from different species. The results provide important insight into how the Smc3-Scc1 DNA exiting gate is opened, how Pds5 and Wapl maintain the opened gate, how Pds5 and Scc3/SA recruit different cofactors, how Eco1 and Sororin antagonize Wapl, and how Scc2/Nipbl interacts with Scc3/SA. The models are for the most part consistent with published mutations in these proteins that affect cohesin's functions in vitro and in vivo and raise testable hypotheses of cohesin dynamics and regulation. This study also serves as an example of how to use AF to build models of protein complexes that involve the docking of flexible regions to globular domains.

Major points
(1) As it stands, the manuscript is simply too long and not readable. The authors should streamline their presentations and remove excessive speculations and models of minor importance.

(2) AF has been accurate in predicting both the fold and sidechain conformations of globular domains. It is less accurate in predicting structural regions with conformational flexibility. Comparisons of predicted and determined structures of large protein complexes have shown considerable differences, particularly with respect to regions lacking tertiary fold. The authors should be more cautious in interpreting some of their models, particularly when the predicted models are inconsistent with determined structures and published biochemical data. For example, human WAPL-C in isolation does not interact with the SA-SCC1 complex while the N-terminal region of WAPL does.

(3) The predicted SA/Scc3-Pds5-Scc1-WaplC quaternary complex is fascinating. Can the authors provide some experimental evidence to support the formation of this quaternary complex or at least the formation of the SA/Scc3-Pds5-WaplC ternary complex? In vitro pulldown or gel filtration can be used to test their predictions.