Did it have to be this way? I mean all of it – biochemistry, the molecules of life. More specifically, as proteins evolve and change, how many paths could they have taken that would have taken them to the same sorts of function?
That’s a pretty hard question to answer, since we’re looking at a billion years of evolutionary tinkering, driven by all sorts of random craziness and a willingness to run with anything at all that works. But here’s a new paper from a team at the University of Chicago that tries to address the question empirically. They’d previously worked out a plausible sequence for the ancestral steroid receptor, from whence all the others have descended. It’s selective for the estrogen-response-element in DNA, so that was the original mode of action. Way back when, three particular mutations in the DNA recognition region of the protein sent some of them down the track for selectivity to the steroid-response-element (including androgen response elements and others), which is still the major divide in the mechanistic details of the whole family.
In this paper, the group systematically examines mutations at those three key sites (and one adjacent one that also varies in the superfamily). A library of all 160,000 mutants was prepared, and they used yeast fluorescent reporter assays to figure out the DNA binding and transcriptional competency of the whole shebang. As an aside, I really love modern molecular biology and chemical biology, because you can actually do experiments like this – just make every single one of the mutant proteins, test them all, sequence the ones that you find interesting, and nail down the answer. It’s not easy, and it’s not an afternoon’s work, but I well remember the days when a proposal like this would have been considered dangerously wasteful and stupid.
So starting from ancestral ERE selectivity, they found 828 variants that were specific for SRE DNA sequences, working as well as the current steroid receptors or better, with no affinity for the ERE sequences. Interestingly, they get to the selectivity in different ways, with different amino acid/DNA contacts than are seen in nature. Graphing these out to see what the relationships were, it appears that virtually all of the functional mutants (1351 in total, including unnatural ERE selective ones) can be reached by single-point mutations from each other without going through inactive mutant proteins. The new SRE-specific protein sequences are scattered all around the graph; there’s no one bottleneck mutation that they particularly have to pass through.
And this persists under the constraints of many evolutionary models. Even if you insist that every step has to yield better affinity for SRE sequences, there are still many ways to get to them. In fact, the SRE sequences that we have now represent neither the shortest mutational path nor the highest-affinity final results. Over 90% of the SRE-selective mutants can get there by a path that’s no longer than the historical one. The evidence is that they just happened to happen that way, they worked, and here we are. It could have been many other ways, and if you reset they clock, odds are that something else would have fit instead. Another result from the graph is that if you start from different ERE-selective points, you have different paths to new SRE-selective ones. So the starting ancestral sequence we happened to have also was an influence; if you wound the clock back even further before the evolution of the ancestral receptor itself, you’d have a whole possible landscape of both forms.
There’s a further complication, though. There are eleven mutations (11P) that also differ between the ERE- and SRE-selective proteins, although they’re not directly at the DNA binding site. If you take those 11P mutations out and make the 160,000 variants again at the four mutation sites around the DNA-binding helix, things are more constrained. Now there are only 43 ERE-specific ones and 41 SRE-specific ones in the whole batch: 99.92% of the mutants are just nonfunctional, and what’s worse, the functional ones are almost entirely connected through nonfunctional nodes. So the ground had to be prepared – the relatively permissive protein landscape isn’t always there, and what we’re seeing is very likely the result of something like that being in place for the DNA-binding mutational landscape to be explored productively in the first place.
So how and why did the 11P permissive mutations take place? Looking at the mutational landscape, it fits with the hypothesis that these nonspecifically increased the affinity of the protein(s) for both ERE and SRE DNA sequences, and seem to have been part of a general improvement in the function of the steroid receptors. These general increases in affinity opened the landscape up to new functional mutations. Here’s the authors’ summary:
Our results shed light on the roles of determinism and chance in protein evolution. The primary deterministic force is natural selection, which drives the evolution of forms that optimize fitness. Chance appears in two non-exclusive ways: as historical contingency, when the accessibility of some outcome depends on prior events that cannot be driven by selection for that outcome; and as stochasticity, when there are paths to numerous possible genotypes of similar function, and which one is realized is random. . .Our results point to strong stochasticity and contingency in the many histories by which SRE specificity could have evolved. Hundreds of genotypes encoding SRE specificity were accessible from AncSR1, but selection for that function alone could not have deterministically driven evolution down any of those paths, because all were contingent on permissive mutations. . .
So the role of chance in evolution is there to see: chance built on chance, actually, with all the time in the world (well, all the time in our own world) to try things out. Things may look like they evolved deterministically, because we tell ourselves stories and our brains themselves seem to have evolved to look for them. But in reality, it’s likely that a lot of molecular-scale evolution just happened for no particular reasons at all. . .
Note: All opinions, choices of topic, etc. are strictly my own – I don’t in any way speak for my employer