A single residue dictates a fold

Anfinsen’s dogma — that the amino acid sequence of a protein uniquely determines its structure — naturally leads one to the idea that identity between amino acid sequences means identity between structures. This has proven to be a successful paradigm: sequence similarity reliably predicts structural and functional similarity. Evidence accruing in recent years, however, suggests that for small proteins, at least, this assumption may not be entirely safe. Adding to this view, in an article in PNAS this week (it’s open access, so open it up), a research team led by Philip Bryan reports that they are able to generate significantly different folds with divergent functions from sequences that differ by a single amino acid.

The researchers in this study took two independently folded domains from streptococcal protein G that have different structures and sequences, called G_A and G_B, which bind human serum albumin (HSA) and part of an antibody (IgG), respectively. As you can see from several figures in this paper, G_A has an all α-helical structure, while G_B has a helix and a four-strand β-sheet. In previous papers they have undertaken a process of slowly mutating these two domains towards each other, most recently generating a pair of proteins they called G_A88 and G_B88 (which I have mentioned before) that retained their original structures and functions despite differing in their amino acid sequence at only seven places (i.e. they were 88% identical). Beyond this point, mutating the sequences towards each other causes the protein stability to degrade. However, they manage to complete the project by generating sequences that are 91, 95, and 98% identical while retaining much of their original function.

G_A98 and G_B98 differ only in the identity of the amino acid at position 45, which is leucine in G_A98 and tyrosine in G_B98. The tyrosine seems to be critical in stabilizing a hairpin turn in the G_B structure, possibly by interactions with an adjacent aromatic residue (F52 – see Fig. 6). In the structure of the G_A95 protein (Fig. 5), L45 is partially solvent exposed, and therefore probably does not contribute substantially to stability. Of course, as a leucine is also hydrophobic, one might expect that it would also interact favorably with F52. This seems to be the case, as the G_A98 protein binds to both HSA and IgG, suggesting that it transiently samples the G_B structure.

As with studies I have talked about previously on lymphotactin, which has two structures in its native state, and the cro proteins, which can have different structures despite similar sequences, this research indicates that the relationship between sequence space and fold space may not be as straightforward as previously believed. We have known for some time that distance in sequence space does not imply distance in fold space — the convergent evolution of numerous proteins has shown us that, at least. These experiments inform us that distance in fold space need not imply distance in sequence space, that it is possible for a protein to undergo a structural saltation in which a single mutation gives rise to a dramatic difference in structure.

It remains to be seen whether this is true in larger proteins as it seems to be in small proteins and domains. If it is generally the case that proteins can drift towards new folds without losing their native function, then “jump” over to a completely different structure, without transiting the molten-globule state, then our understanding of evolution, particularly early evolution, may change significantly. For instance, the objection that proteins could not evolve new functions without wandering into the unfolded state loses its weight. Moreover, given that in two of these cases the proteins actually sample both structures (and hence, both functions), it is not even necessarily true that a protein must lose its original function before evolving a new one. Equilibrium switching between structures would allow a protein to fulfill both the old and new functions, albeit likely at reduced efficiency. As I’ve mentioned before, one answer to this lack of efficiency would be altering protein concentration by adjusting gene dosage, i.e. gene duplication. From that point, the generation of fold and functional diversity by evolution might be a straightforward process.

Alexander, P., He, Y., Chen, Y., Orban, J., & Bryan, P. (2009). From the Cover: A minimal sequence code for switching protein structure and function Proceedings of the National Academy of Sciences, 106 (50), 21149-21154 DOI: 10.1073/pnas.0906408106 OPEN ACCESS