Two folds for lymphotactin

While reports of my man-crush on Brian Volkman are in general much exaggerated, it is true that I adore one of the systems he studies, the bizarre chemokine lymphotactin. In case you couldn't guess from past posts here, I am endlessly fascinated by this protein, and so I was very happy to finally see his latest paper on the subject in today's feed-dump from PNAS. Previously published research out of Brian's group indicated that lymphotactin adopted two totally different structures under different solution conditions. The new paper provides high-resolution structures of the non-chemokine fold and demonstrates that the structures have distinct activities, both of which are essential for full lymphotactin function in vivo.

Lymphotactin is a chemokine, a small protein which has the property of binding to molecules in the extracellular matrix (ECM) such as long polysaccharides, and also of activating certain G-protein coupled receptors (GPCRs). A previously-solved structure of lymphotactin (left) displayed a normal chemokine fold (explore this structure at the PDB), which is designated Ltn10. However, in order to get this structure by NMR, the Volkman lab had to either engineer in a second disulfide bond (as in this structure), or acquire their data under very specific conditions (10 °C, 200 mM NaCl). The reason for this is that at under reasonable biological conditions (37 °C, 150 mM NaCl), their spectra showed evidence of an alternate conformation. At higher temperature and lower salt, the peaks corresponding to the structure at left disappeared entirely and were replaced by peaks corresponding to an alternate conformation.

You can see that alternate conformation, called Ltn40, at right (explore this structure at the PDB). As you can see, this structure is dimeric, taking the form of a β-sandwich. The β-sheets themselves have a sort of Greek key meander arrangement. You can learn more about β elements, protein topology, and protein structure at Larry Moran's Sandwalk. This new fold buries a substantial number of hydrophobic side-chains between the β-sheets. At the same time, a preponderance of positively-charged residues is exposed to solution. The α-helices of the chemokine structure appear to unfold completely, a new β-strand forms at the N-terminus, and the existing sheets shift their hydrogen-bonding register by one residue, meaning that β1 and β3 are rotated 180° along their lengthwise axes.

The existence of two native folds under reasonable physiological conditions (K_eq near one at 37 °C) poses two questions. The first is whether and how fast the structures interconvert. The fact that the equilibrium of a given sample can be shifted with temperature and ionic strength implies that this is an active equilibrium, i.e. that the energy barrier is low enough to cross using just thermal energy. Tuinstra et al. used an NMR experiment to establish that interconversion takes place on a timescale of about 100 ms.

The second question that naturally comes to mind is whether the two structures have different functions, and what those functions are. With a few clever experiments in which they used mutations to stabilize one fold or the other, Tuinstra et al. demonstrated that only the Ltn10 fold activates the partner GPCR, and only the Ltn40 fold binds to heparin, a polysaccharide often found in the ECM. Thus, neither fold possesses the full range of biological functions of lymphotactin; in order to fulfill its biological role it must switch between these structures in vivo. Moreover, the exclusivity of these functions between folds naturally suggests a switching mechanism for regulation.

This is an interesting and important finding because it is (so far) the only example of a protein adopting two completely different stable folds with no hydrogen bonds in common at equilibrium. Trivially, natively disordered proteins adopt multiple conformations under physiological solution conditions, and many proteins alter their conformations in response to ligand binding while keeping most of their hydrogen bond network intact. In this case, however, an existing network of stabilizing bonds is completely disrupted in order to form a new fold with a totally different function. I've already discussed some of the implications of this with respect to protein folding, and in regards to the recent transitive homology studies out of the Cordes group. Lymphotactin offers lessons and ideas for protein folding and evolution that must be taken into account. In particular, the fact that point mutations can significantly stabilize one or the other of these structures implies that there may be previously unsuspected shortcuts through structural space between folded states that avoid unproductive or energetically unfavorable molten globules.

In addition, these results signify that the Anfinsen paradigm that dominates our understanding of protein structure ought not be taken for granted. In many cases it is true that a peptide sequence uniquely determines a single structure under all physiological conditions. Of course we have known for some time that certain peptide sequences do not produce ordered structural ensembles at all. What the lymphotactin example makes crystal clear is that a given sequence can yield an ensemble with multiple energetic minima that reflect related but topologically distinct structures. Tuinstra et al. suspect that this phenomenon has not been noted previously because structures of this kind would not be amenable to crystallization, or would only crystallize in one (of many) structures. If this is so, then as more and more proteins are studied using solution techniques under physiological conditions we may find multiple structural minima in a variety of proteins. Such discoveries may significantly enhance our understanding of the protein regulation, function, and evolution.

1. Tuinstra, R.L., Peterson, F.C., Kutlesa, S., Elgin, E.S., Kron, M.A., Volkman, B.F. (2008). Interconversion between two unrelated protein folds in the lymphotactin native state. Proceedings of the National Academy of Sciences 105 (13) 5057-5062. DOI: 10.1073/pnas.0709518105

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The Evolution of Protein Folds

Now online for next week's edition of PNAS is a commentary by Alan R. Davidson (1) about a paper in this week's edition of PNAS out of Matthew Cordes' group (2). Both are worth reading because they speak to a very interesting question: where do new protein folds come from?

The Roessler et al. paper doesn't address this question directly. Their initial intention was to identify the relationship between two distantly homologous proteins: P22 Cro and λ Cro. Though they both belong to the Cro repressor superfamily, these two proteins have just 25% sequence identity and significant dissimilarities in structure, as you can see on the right in the figure I have shamelessly stolen from the paper. P22 is an all α-helical structure that appears to be exclusively monomeric, while λ is an α/β structure that forms a dimer with nanomolar affinity. In an effort to bridge the structural gap between these proteins, Roessler et al. looked at group of proteins related by transitive homology. The idea, as they put it, is that

In this approach, two dissimilar sequences, A and C, are indirectly linked if a third "intermediate" sequence B exists with sufficient similarity to both A and C to imply homology with both proteins. The relationships between A and B and between B and C combine to support distant common ancestry between A and C.

Thus, they identify 3 intermediates, each with about 40% identity to its nearest neighbors, that bridge the sequence gap. Then they ask whether these sequence intermediates are also structural intermediates.

The answer is "yes", and in a somewhat surprising way. It is not the case that each step along the transitive pathway slightly increases β-strand content. Rather, while Xfaso 1 has an all-α structure very similar to P22 and is a monomer, Pfl 6—which is 40% identical (!)—has an α/β structure similar to that of λ Cro and dimerizes with ~1 mM affinity. This dissimilarity allows the authors to present some interesting ideas about the evolution of the Cro family which are summarized in their Figure 4. But what seizes Davidson's imagination is the conjunction of fairly high sequence similarity with structural dissimilarity. What makes this conjunction even more impressive is that the sequence identity is evenly distributed while the structural differences are not. The N-termini of these proteins contain structurally similar helix-turn-helix motifs, so they primarily differ in the structures of the C-termini. Yet amino-acid identity holds up across essentially the whole sequence.

Why is this such a surprise? Well, there are a variety of reasons, which Davidson outlines pretty well. It boils down to this—for a given sequence, it is generally possible to mutate a significant percentage of the residues without disrupting the fold. That is, the sequence overdetermines the structure. Consequently, proteins that have homologous or significantly identical sequences (and 40% identity would probably fall in this range) are expected to possess very similar structures. This poses a problem for protein evolution because it is expected that the initial pool of folds was rather small. If protein folds are highly resistant to disruption or alteration by mutations, it's difficult to imagine how the present enormous diversity of folds arose.

This impression is actually somewhat mistaken. It's typical to perform X→Ala mutations in these studies, and while this can occasionally produce significant cavities in a structure, it probably significantly underestimates the potential effects of a mutation at any given spot. For buried residues, size increases and the introduction of unbalanced charges (X→Trp, Asp, Lys, etc.) are mutations likely to drive the formation of new structure. For solvent-exposed residues, the introduction of bulky nonpolar side chains (X→Phe, Leu, Ile, etc.) would also be more likely to result in a novel fold than the typical approach. I have a feeling that these kinds of mutations are significant in this context, but I cannot check this because both 3bd1 and 2pij are still on hold and cannot yet be retrieved from the PDB. I may elaborate on this point when the coordinates are released to the public. For the time being I should point out that though the quantity of identity is similar between the two termini is similar, the quality is not: identical residues in the N-terminus almost all appear together, while in the C-terminus they are spread out. However, it is worth noting that what groupings of identical residues can be found in the C-terminal region tend to occur within the structural features that changed.

Davidson bears out this point when he refers to some experiments that have shown that a few mutations in key spots could change a protein's fold significantly. He seems to be unaware, however, of natural instances in which highly similar sequences produce dissimilar folds. As I have mentioned before, the upper limit on sequence identity producing dissimilar folds is known. Structural studies that Brian Volkman's group published in 2002 (3) demonstrated that the maximum sequence identity that allows for the adoption of a completely different structure is 100%. That is, given reasonable changes in solution conditions a single peptide sequence can produce two entirely different folds.

The last time I blogged on lymphotactin I discussed the implications of Brian's findings for the protein folding and protein structure prediction crowds. In the context of sequence similarity, however, the lymphotactin story also has implications for evolution as well. To a certain extent it suggests that we have been somewhat blinded by Anfinsen's dogma, in particular the assumption of the unchallenged minimum. The lymphotactin result indicates that context can be extremely important—a sequence that stably folds into one structure in one set of conditions will not necessarily maintain that structure under different conditions. In an elementary sense, we know this already, since we are aware that high temperatures and high concentrations of cosolutes such as guanidine and urea tend to unfold proteins. The important idea is that conversions between Anfinsen-like folds (i.e. folds that conditionally dominate the energy landscape) can occur within the range of conditions that can be achieved physiologically. Because the relationship between sequence and structure is not truly one-to-one, fold diversity may be much easier to achieve than we have suspected on the basis of existing structural studies.

In the end, the results of Roessler et al. provide a powerful counterpoint to conventional expectations about the relationship between sequence identity and structural homology. It appears that Cro proteins group into two kinds of structures, underscoring the well-known stability of protein folds to mutation. However, the structural discontinuity, not hinted at by sequence comparison alone, reinforces the point that fold diversity may be significantly easier to achieve than alanine-scanning mutagenesis experiments have led us to believe. Davidson nonetheless still has a significant point. In this case, it appears that transit to the new structure was relatively short, and Cro is functionally a dimer in both forms—Xfaso 1 Cro forms a dimer in the crystal, and all Cro repressors are expected to dimerize on DNA. But what happens in the transit to a completely novel structure? Can function be maintained as the protein navigates the molten-globule strewn sequence space between stable folds, and if so, how? These are questions that we will have to answer as we develop a greater understanding of the evolutionary history of biomolecules and de novo protein design.

1. Davidson, A.R. (2008). A folding space odyssey. Proceedings of the National Academy of Sciences, 105(8), 2759-2760. DOI: 10.1073/pnas.0800030105
2. 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
3. Kuloglu, E.S. (2002). Structural Rearrangement of Human Lymphotactin, a C Chemokine, under Physiological Solution Conditions. Journal of Biological Chemistry, 277(20), 17863-17870. DOI: 10.1074/jbc.M200402200 OPEN ACCESS

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It came from the Protein Society! (Part One)

I went to the annual Protein Society symposium a few weeks ago and have finally had some time to organize my thoughts about it, so I figured I'd put some of them up here. Expect a couple of these to show up.

One of the most interesting presentations at the Protein Society (besides my own scintillating poster on field-cycling, haha) was Brian Volkman's poster on lymphotactin. This is a really interesting story that somehow seems to keep flying beneath the radar of most people, but intellectually it represents a giant challenge.

In a nutshell, the story is this: lymphotactin is a small signaling protein of the chemokine family, a group of proteins that are important for various kinds of regulation, including in inflammation and disease. Under fairly standard experimental conditions (200 mM NaCl, 10 °C) the protein adopts a normal chemokine fold, but at 45 °C (for reference, body temperature is 37 °C) and low salt, it takes on a totally different fold. You can read the original paper on this here. Of course, when you see something like this it's natural to ask what the physiological relevance of the finding is. Brian's poster at Protein Society basically answered this question by illustrating different biological roles for the two forms. The short version is that the conformational change appears to be some sort of regulatory switch. I'll have more on that in the next episode.

What I want to talk about here was what wasn't said about this at the meeting. After all, we were forced to witness the usual ninny-argument over whether folding was a linear pathway or a funnel of some kind. While it was refreshing to hear a lot more people pointing out that this distinction is more or less meaningless, it's odd that nobody is tackling the question through a protein like lymphotactin. Consider the following experiment: perform phi-value analysis or GdHCl-dependent HX experiments on lymphotactin to find what portions of the protein are structured in the folding transition state. We can imagine two outcomes.

In the first, the transition state is found to be completely different; that is, residues with high phi-values or the last residues to lose protection in the HX experiment are completely different for the two conformations. This would suggest that the latest common intermediate is the random coil (RC), and that the very first move towards a folded state dictates the state one finally arrives at (N1 or N2). Any intermediates (I1 and I2) along the pathway are unique to the end state, rather than shared between the two conformations (see right). This would fit most closely with the pathway view promulgated by Englander. Given that the hydrogen-bonding patterns are totally different for the two conformations, this might be expected.

On the other hand, it's possible that a residue or cluster of residues have similar phi-values, or lose their protection at a similar GdHCl concentration, between the two conformations. This would not be completely probative, as the similarities could quite easily be restricted to the observables and represent two different underlying structures. However, if veridical this might suggest that the latest common intermediate lies somewhere other than in the random coil. This would be more similar to the funnel view, in which a conformational search over the outcomes available to an intermediate gives rise to the ultimate choice of native structures. I've cartooned the idea over to the left. In this case I* represents a partially-folded intermediate that selects an end state based on the conditions.

After all, lymphotactin in both its native forms exists in physiological extracellular conditions. Knowing whether the protein must pay the full energetic cost of completely unfolding, or if it can switch conformations by taking a less-costly move to a common intermediate may be of significance to understanding the biology. And while these two alternatives (like the underlying models) are not as different as they may seem of first blush, the answer may do much to distinguish whether the conformational search of the funnel model or the deterministic folding of the pathway model is the best representation of the folding process.

Another major implication here is for the protein structure prediction crew. After all, the CASP-type experiments are all geared to the idea that a given sequence should give rise to a single folded structure. Lymphotactin is a clear counterexample to this idea, and while it may be unique, we certainly don't know nearly enough about proteins to let the dogma go unquestioned at this point. This makes for a much larger computational problem. I'm not a huge expert on these experiments, but my impression is that the programmers don't concern themselves too much about the characteristics of the solution the proteins are in. The assumption that co-solutes don't much matter is vastly simplifying, but as Brian's work shows, may ultimately limit the predictive power of these algorithms in significant ways.

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