Why are Xfaso and Pfl Cro so different?

A few weeks ago, when I posted on the transitive homology studies performed by the Cordes group, I promised a closer look at the structures when they became available. If you'll recall, one of the central findings of the Roessler et al. paper (1) was that the Xfaso and Pfl 6 Cro proteins, though they had 40% sequence identity, as well as an identical function, had very different structures and dimerization characteristics. The Pfl6 Cro structure is now available in the PDB, and Dr. Cordes was kind enough to send me the Xfaso Cro structure, which has been held up by some technicalities. I made the overlay of the structures to the left, with Xfaso in dark green and Pfl 6 in crimson. As you can see, the N-terminal helix-turn-helix motifs of the two molecules overlay very precisely, with some slight differences in orientation in the context helices. The C-terminal portions, of course, are completely different. How did they get to be this way?

Well, I have a few thoughts. To achieve a significant change in structure like we have here, two possibilities suggest themselves. We can destablilize one structure, or we can stabilize the other. So let's try to look at this from both angles. First, the Xfaso structure, which is on the left. The ribbon is orange for residues that don't change between the two proteins, green for mutated sites, and red over a deleted range. I've also drawn in a couple of the mutated side chains that might have an effect. For instance, at the upper right you can see a glutamate of Xfaso Cro that becomes a glycine in Pfl cro. The presence of the glycine may destabilize the helix. Lower on the helix, a solvent-exposed arginine becomes a hydrophobic leucine in Pfl; likely the structure will change to reduce the contact of the leucine with water. By the same token, a partially-buried threonine at the base of the helix gets mutated to glutamate. Not only might this put an unsolvated negative charge inside a hydrophobic region, but favorable helix-capping interactions of the threonine might be broken. Roessler et al. also point out that a pair of cysteines in the Xfaso structure are in a favorable position to form a disulfide bond; both are absent from the Pfl6 sequence.

On the other side of things, what mutations are stabilizing the new fold of Pfl 6 Cro? Check out the image below, color-coded the same way as the previous structure:
There are a couple of things going on here. First, the hydrophobic residues. The leucine that was an arginine in Xfaso Cro has indeed been buried, up at the top of the structure. Nearby, the glutamate that was a partially-buried threonine in Xfaso is now fully solvent-exposed. In the same vein, an aspartate has become an isoleucine near the middle. This new isoleucine appears to pack into the core of the companion protein near a conserved isoleucine (and a new methionine), and probably accounts to some degree for the stability of the dimer. There are a wealth of minor effects too—an alanine to arginine mutation has created a charged group that is solvent stabilized on one of the strands; down near the bottom a proline-to-arginine change probably has allowed the extension of a helix. Some other side-chains are drawn that I was taking a look at, but probably aren't critical, and of course I've probably missed a few that matter.

The R→L, T→E, and D→I mutations probably do a great deal to change the most stable conformation from all-α to α/β and strengthen dimerization in solution. However, just from looking you can never be sure what mutations are the most important. Doubtless the Cordes lab is already examining which residues are critical in altering the conformation. It will be interesting to see whether point mutations tend to move Xfaso from the all-α to the mixed structure, or whether they produce molten globules.

1. Roessler, C.G., Hall, B.M., Anderson, W.J., Ingram, W.M., Roberts, S.A., Montfort, W.R., Cordes, M.H. (2008). Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds. Proceedings of the National Academy of Sciences, 105(7), 2343-2348. DOI: 10.1073/pnas.0711589105

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What are you keeping that for?

Analyzing the conservation of DNA and protein sequences has proven to be an incredibly valuable approach in structural and molecular biology. The fundamental principle underlying the use of conservation data is the idea that conserved sequences are invariant for functional reasons. Changing the amino acid or base at these conserved sites, it is assumed, would lead to a catastophic structural disruption of a protein, eliminate an important functional group, or (in the case of DNA elements) substantially distort expression levels or splicing. If this is so, genetic elements that are ultraconserved (>200 bp segments that are 100% conserved between human, mouse, and rat genomes) should be positively essential to the survival of an organism. However, a recent study published in PLoS Biology (open access) indicates that removing at least some of these ultraconserved elements (UCEs) has little or no effect on development or longevity in mice.

Ahituv et al. knocked out four ultraconserved elements in C57BL/6J mice, none of which are known to code for protein. This is normal; more than half of these ultraconserved regions (481 have been identified) are not transcribed into RNA. It's thought that these non-coding regions are important for gene regulation, and some preliminary experiments suggested that this would be the case for these four. But homozygous or hemizygous (some of these genes were on the X chromosome) knock-out mice developed normally, for the most part, and were born in roughly the ratio one would expect from a Punnett square. Gender ratios and litter sizes were also normal -- this is particularly compelling in the case of the 2 UCEs located on the X chromosome. If losing these UCEs were fatal or even mildly problematic, we would expect to see some evidence of this in the gender ratio of the pups.

Element deletion was not entirely without effect. Knocking out one of them caused a very low incidence of male mice born with only one kidney. Another had a slight effect on the expression of a gene called Sox3 in the developing brain. However, the genes flanking these deletion sites have much more serious effects when they are deleted or otherwise disrupted -- several of these cause embryonic lethality. So the UCEs have enormously reduced effects compared to the flanking genes.

If they're not important, why are these regions conserved? That's a tough question, and in a way hinges on a false impression. This particular disruption of these regions did not adversely affect an extremely inbred pool of mice that were well-fed and kept in controlled climates. That is, these organisms are genetically abnormal and face little stress. Their responses to these mutations may not be indicative of mice in general, or even of other strains of laboratory mice. Moreover, these regions may only be important in stress conditions that are nonetheless ordinary -- development under conditions where some nutrient is absent or rare, for instance. Or the effect may only be felt after many cumulative generations.

Another possibility is that of redundancy or subtlety of phenotype. Some other element may be acting to do the job once performed by these UCEs. There is a similar story with growth factors, expressed proteins that are essential but nonetheless seemed not to produce a phenotype in mice unless several were knocked out of the genome simultaneously.

And finally, there is a possibility that the reason UCEs do not mutate is not that they are essential but that they can easily gain function that is fatal. That is, mutations turn the UCE into a toxic piece of DNA, but some minor function keeps them in the genome. This interpretation is not as well supported by existing data, however, which suggest that it is specifically the loss of these elements that reduces fitness.

As usual, this interesting work raises more questions than it answers. Understanding the reasons why UCEs are preserved with such high fidelity through various species will likely contribute a great deal to our understanding of expression dynamics and evolution.

Ahituv N, Zhu Y, Visel A, Holt A, Afzal V, Pennacchio, L. A., Rubin, E. M. "Deletion of Ultraconserved Elements Yields Viable Mice" PLoS Biology Vol. 5, No. 9, e234

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