1. Reviewer #3 (Public Review):

    Evolution is a historical phenomenon that plays out over time through the complex interaction of the stochastic processes of mutation and genetic drift and the deterministic process of natural selection. Biology has seen a vibrant debate over the last few decades over what this means for the repeatability of evolution, and to what degree evolutionary outcomes are shaped by the combination of necessity, chance, and historical contingency. This debate has led to intense empirical study of these factors in evolution. Reconstruction and examination of functional protein evolution has been one of the cleverest and most interesting systems used in this study. Here, the authors seek to examine the roles of chance, contingency, and necessity in the evolution of protein-protein interactions (PPI) between BCL-2 family proteins and their coregulators. They specifically look at the evolution of specific interaction between BCL-2 and BID and the more generalized interaction between MCL-1 and coregulators BID and NOXA. They authors reconstructed the last common ancestor protein of BCL-2 and MCL-1 and a series of intermediates along their respective lines of descent. They then used a very clever Phage Assisted Continuous Evolution (PACE) system to subject replicates from each time point to selection for different PPIs and examined variation in sequence variation. By looking at evolution in replicates from different time points, they were able to disentangle the effects of chance, contingency, and necessity. They found that necessity played little role in protein evolution, with little predictability between replicates of single time points and among those from multiple time points, indicating that there was no single pathway through sequence space to the selected function. They did, however, find strong and synergistic effects of chance and contingency. They did tests to demonstrate that the effects of contingency were due to epistatic interactions that affect the viability of particular historical paths. Chance, meanwhile, had effects because multiple mutations could lead down paths to the selected function. The authors conclude that history and chance must be considered when attempting to understand protein function evolution, and that the sequences of proteins with given functions reflect do not reflect necessary pathways or constrained endpoints, but particular and idiosyncratic histories. Moreover, they suggest that contingency may need to be considered as a fundamental aspect of the evolutionary process, along with mutation, drift, and selection.

    Altogether, this is a wonderful and interesting manuscript that makes a substantial and material contribution to our understanding of how history and chance affect evolution. It even speaks to the nature of more fine-grained protein sequence evolution relative to neutral and adaptationist theories. The amount of work and thought that went into the research is nothing less than astonishing. Every time I found myself wondering, "but did they check this...", I found that they, in fact, did in the next section. The work is solid, and the results are robust. I do not see anything that concerns me in the nitty gritty of the actual scientific work. I do, however, think that the authors should engage the work that philosophers of science have done in the last decade or so to better develop our conceptual understanding of contingency and reconsider the meaning of their findings in light of that work.

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  2. Reviewer #2 (Public Review):

    The extensive description of mutational paths using high-throughput phenotyping combined with sequencing provides a rich and useful data set. However, the experimental setup has some serious limitations.

    First, the authors want to address the evolution of protein-protein interactions, but they actually do so comparing the interaction of actual and ancestral proteins with actual human BID and NOXA proteins. The analysis would have been stronger with reconstruction of ancestral sequences also for the BID and NOXA proteins, to test interaction of two proteins at the same evolutionary node. Actually, characterization of protein-protein interactions between proteins from Trichoplax, for example, suggest that the results may be different (Popgeorgiev et al., Science Advances 2021).

    Second, the specificity of the binding of NOXA to MCL-1 and not to BCL-2 seems to be an artifact due to the use of peptides instead of full-length protein during interaction assays. This is explicitly indicated in one of the reviews the authors cite in their introduction (Kale et al., 2018, p67). This review mentions a JBC paper clearly demonstrating that BCL-2/NOXA interaction do occur even in human cells: Smith AJ, Dai H, Correia C, Takahashi R, Lee SH, Schmitz I et al. Noxa/Bcl-2 protein interactions contribute to bortezomib resistance in human lymphoid cells. J Biol Chem 2011; 286: 17682-17692.

    Third, the same review also stresses that these proteins are partially membrane-bound in vivo. So testing their interactions in soluble protein bioassays is far from physiological relevance. Actually, such a warning appears already in one of the bullet points from the Kale review:

    "The majority of studies examining the interactions between BCL-2 family proteins use truncated proteins or peptides of the BH3 region at physiologically irrelevant concentrations or in the absence of membranes leading to confusion in defining the core mechanisms of the BCL-2 family proteins."

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  3. Reviewer #1 (Public Review):

    This manuscript reports a novel and original approach to examine the possible mutational paths underlying directed protein evolution.

    The authors conclude from their experiments that "Necessity was almost entirely absent" (line 209). Indeed, the vast majority of states evolved in just one replicate from one starting point. But this is the problem of a half-full glass: is it half full or half empty? If I understand Fig.4F correctly, one can still detect amino acid changes that recapitulate historical substitutions, and others that revert to the historical state, so it does not seem that necessity is "almost entirely absent". Furthermore, several of the amino acid changes that were detected may not have any effect on NOXA or BID biding, maybe they occurred because of mutational bias, drift or hitchhiking. If this is the case, then one cannot compare all acquired states in each trajectory and conclude about the importance of chance, as in this sentence for example: "Pairs of trajectories launched from the same starting point differed, on average, at 78% of their acquired states, indicating a strong role for chance" (line 219). There are causal mutations that arose repeatedly during PACE replicates from each starting genotype and these mutations do indeed confer the selected-for specificity in their "native" background (as is nicely shown in Figure 6A-B). So this, to me, is evidence for necessity.

    Loosing a binding property can probably occur via multiple ways, which are likely to be more numerous than gaining binding for a given protein. It would be nice to discuss this point in more detail.

    The experiments presented are limited to one protein family and to the binding properties to two different proteins. In living organisms, each protein is likely to exhibit particular properties such that it can bind or not bind to hundreds of different proteins, and not just two as tested here. So the constraints present in living organisms may be much larger than the ones present within this experimental evolution set up. Furthermore, the tested proteins probably encounter other constraints in their native environment besides affinity for other proteins, and it is yet unclear whether the variant forms obtained here via experimental evolution would be fine to replace the endogenous proteins in living organisms. It is therefore difficult to generalize from the obtained results to all types of evolutionary changes. In general, the conclusions should be toned down and focused on this particular example.

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  4. Evaluation Summary:

    This manuscript, which will be of interest to students of evolution and anybody interested in protein function, uses an original, clever, high throughput, and rapid experimental protein evolution method to assess the roles and contributions of contingency, chance, and necessity in the evolution of protein-protein interactions. The authors focus on the animal BCL-2 protein family and on the evolution of their binding properties to two proteins, NOXA and BID. Using several replicates and several starting points, they found little predictability between replicates of single starting points and among those from multiple starting points, indicating that there is no single pathway through sequence space to the selected function, and that historical contingency is the primary cause of protein evolution here. The presented results convincingly illustrate the potential of this novel technology for future work in directed protein evolution.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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