Douglas Axe, Protein Evolution, and Darwin's Doubt: A Reply
Editor's Note: For a response to Doug Axe (see here and here), we are pleased to welcome Martin Poenie to our pages. Dr. Poenie is Associate Professor in Molecular Cell & Developmental Biology, University of Texas at Austin.
Douglas Axe and Stephen Meyer want us to believe that protein evolution is impossible and that Axe's experiments somehow inform that belief. His experiments, he claims, take the approach of a chemist attempting to make the minimal amount of substitutions needed to transform specificity of one enzyme into that of another similarly structured enzyme. In my view, the experiments have little to say about evolution and are singularly uninformative. His experiments have also been widely criticized.
As Axe notes, there are 250 out of 381 amino acids that differ between BioF and Kbl such that the two sequences are 34% identical over 381 aligned positions. From an evolutionary perspective, this is a long distance. It is not surprising that his efforts to swap functionality by swapping a few residues failed. Approaches similar to what Axe has done have failed repeatedly. As noted by Harms and Thornton, "The reason studies of this type fall short is that they ignore history" (Harms and Thornton, 2010, Current opinion in structural biology 20: 360-366). The Young Earth Creationist Todd Wood summarized the issue succinctly:
Instead of ancestral reconstruction, Gauger and Axe focused directly on converting an existing enzyme into another existing enzyme. That left me scratching my head, since no evolutionary biologist would propose that an extant enzyme evolved directly into another extant enzyme. So they're testing a model that no one would take seriously? Hmmm...
I quote Wood as a good example of someone who you would think would be happy to embrace Axe's conclusions if only they were valid scientifically. But they are not.
It is interesting to note that efforts to change enzyme/binding specificity using ancestral reconstruction actually do work. For example:
- Bridgham, J. T., E. A. Ortlund, et al., 2009, "An epistatic ratchet constrains the direction of glucocorticoid receptor evolution.", Nature 461: 515-519.
- Smith, S. D., S. Wang, et al., 2013, "Functional evolution of an anthocyanin pathway enzyme during a flower color transition." Molecular biology and evolution, 30: 602-612.
I find the Gauger and Axe study uninformative (beyond that which we already knew) because they have no idea what their substitutions actually did to their protein. For example, these mutations could have simply led to an unfolded protein. They reject this idea based on a generalization that "it appears that about 10% or more of the residues in natural proteins need to be changed before the cumulative structural disruption can be expected to cause complete loss of function" (Axe, D. D., 2000, Journal of molecular biology 301: 585-595).
Yet in Axe's own words from that paper, "In both experiments, complete loss of activity demonstrates the importance of sequence context in determining whether substitutions are functionally acceptable." The fact is, they do not know what they did to their protein other than to change residues and show (unsurprisingly) that it did not work. This type of experimental design would be cause for rejection from any mainstream scientific journal and perhaps it explains why he published it in his own journal.
In the wider context of protein evolution, I find this fixation on identifying random point mutations as "Darwinism," and then attempting to disprove Darwinism by showing the odds of this or that, very unpersuasive. For one thing, it leaves out a major mechanism for change involving recombination. Recombination can do all the things that Axe thinks are impossible. For example, recombination can generate multiple substitutions in one step and still generate one or two functional proteins as the outcome. Quoting Watson et al. (2011, Evolution, 65: 523):
Whereas asexuals must move against selection to escape local optima, sexuals reach higher fitness peaks reliably because they create specific genetic variants that "skip over" fitness valleys, moving from peak to peak in the fitness landscape. This occurs because recombination can supply combinations of mutations in functional composites or 'modules', that may include individually deleterious mutations. Thus when a beneficial module is substituted for another less-fit module by sexual recombination it provides a genetic variant that would require either several specific simultaneous mutations in an asexual population or a sequence of individual mutations some of which would be selected against.
Furthermore, as noted by Romero and Arnold, "Laboratory experiments clearly demonstrate the benefits of recombining homologous proteins: intragenic recombination generates new proteins that are functionally diverse while still having a high probability of folding properly and functioning" (2012, PLoS computational biology, 8: e1002713). Indeed, due to its conservative nature, recombination can explore a "functional ridge" between two proteins (Drummond et al., 2005, PNAS USA 102: 5380-5385).
Finally, in regard to ORFans, in my view, Axe's argument simply backfires. ORFans are found in all genomes; prokaryotes, eukaryotes, bacteriophages and animal viruses. Remarkably, the recently discovered megavirus "Pandoravirus" has 2500 genes, almost all of which are ORFans. The fact that the number of ORFans tends to be constant from one type of organism to the next in prokaryotes and eukaryotes indicates that they not uniquely associated with the Cambrian explosion and that they are likely formed by mundane genetic mechanisms that operate in all organisms.
In one particularly informative example, Toll-Riera et al. (2009. Mol. Biol. Evol. 26, 603-612) identified 270 primate-specific ORFans. Of these, 70% contained a transposable element. In other cases, where a particular gene appeared to be ORFan, the same organism had a paralogue that did show homology with other organisms suggesting that the ORFan in question underwent rapid divergence. Contrary to Axe and Meyer, the fact that ORFans could represent new genes generated by genetic mechanisms such as transposition really throws a monkey wrench into their arguments.