Locking in new functions

The September 24th Nature came in the mail today and as always with this journal (otherwise I wouldn't pay for it!) is full of interesting stuff. One paper of particular interest is a cool merger of evolution, computational biology, structural biology and protein engineering.

An interesting question in evolution is to what degree are changes reversible. In the simplest case, of purely neutral characteristics, the answer would seem to be largely that they are. However, even a purely neutral change will have a certain probability of reverting. For example, since transversions (mutation of a pyrimidine to a purine or vice versa) are less common than transitions (purine->purine or pyrimidine->pyrimidine), a C->G mutation (transversion) is less likely to return to C than a C->T (transition). Similarly, if a C is methylated but that methylation serves no purpose, the methylation will favor conversion to a T, but the T has no such biochemical slanting to mutate to a C. But even these will be small changes.

But throw in some function, and the question gets more complicated. The question that this paper addresses is a specific receptor, the glucocorticoid receptor. A previous paper by the group showed that the inferred ancestral form was promiscuous, primarily bound some related steroids, but did have some affinity for glucocorticoids. This ancestral form existed in the last common ancestor of cartilaginous and bony fishes but by the time of the last common ancestor for bony fishes and tetrapods (such as us) it had fixed a specificity for corticosteroids. These inferred ancestral receptors are referred to respectively as AncGR1 and AncGR2.

While there are 37 amino acid replacements between AncGR1 and AncGR2, it takes only two of these (group X) to switch the preference of AncGR1 to corticoidsteroids. The change is accomplished by substantially swinging a helix to a new position in the ligand binding pocket (helix 7) Only three more substitutions (group Y) enforce specificity for corticosteroids; make all 5 of these changes and you convert a promiscuous receptor with weak activity towards corticosteroids to one activated only by them. But the interesting kicker is you can't make this second set of specificity-locking mutations until 2 other mutations (group Z) are made. The issue is that the first two X mutations cause a significant structural shift which is not entirely stable; without the stability of the group Z pair of mutations the group Y specificity trio can't be tolerated.

But, there's a kicker. If you engineer the AncGR2 protein back to having the ancestral states for groups X, Y and Z, the resulting protein is non-functional for any ligand. Something is going on somewhere in those other 30 changes. Some further phylogenetic filtering suggested 6 strong candidates and the solution of the X-ray structure of the AncGR2 ligand binding domain (though it turns out the prior homology model of this structure was apparently almost dead on). Five of the candidates (group W) turn out to either be in or to contact that swung helix 7. The structure of AncGR1 had been previously solved and a comparison of the AncGR1 and AncGR2 structures showed that the ancestral (AncGR1) forms at these 5 positions stabilize the ancestral position of helix 7 and the derived (AncGR2) amino acids at these positions actually clash with the AncGR1 positioning of helix 7. Aynthesis of AncGR2 with the ancestral amino acids at groups X, Y, Z and W yielded a receptor whose specificity is very like AncGR1. One group W substitution had a strong enough effect it could imbue the ancestral phenotype even without the other group W changes but some of the other group W changes could be made only in pairs to show an effect. Finally, receptors with the ancestral state for combinations of x, y and z mutations (e.g. combining with Xyz -- AncGR2 for X but AncGR1-like at y and z) and found that any combination with xW is non-functional. AncGR2 with ancestral amino acids at x,y,z & w is not as good a receptor as AncGR1 -- suggesting that at least some of the remaining 25 positions contribute.

So, this is a well-detailed case where evolutionary change eventually blocked the route back to the start. A receptor which made the group X changes could still bind the original ligands but that would be lost once the group Y changes were layered on. Group Y changes were probably preceded by group Z changes which would have made reversion to the original binding specificity unlikely -- and the group W mutations really nail shut the door.

This particular system was a single polypeptide chain. But it is not difficult to see how the concept could extend to other biological systems. Co-evolution of interacting proteins, such as a protein and its receptor, or modification of a developmental system could similarly proceed in a stepwise fashion that ultimately prevents retreat. We are a bit lucky in this case that the evolutionary traces are all preserved where we can find them; it is not difficult to imagine a scenario where part of the ancestral form is lost from all extant lineages and therefore invisible to our current vision.



Bridgham JT, Ortlund EA, & Thornton JW (2009). An epistatic ratchet constrains the direction of glucocorticoid receptor evolution. Nature, 461 (7263), 515-9 PMID: 19779450

I'll probably add to my spam issues by pointing this out, but this