Jun 022009
 
ResearchBlogging.orgVaccination plays such an important role in our seasonal influenza strategy in part because we don’t have many medicines that can be brought to bear on the disease. The neuraminidase inhibitors (specifically Tamiflu) are widely stockpiled, and continue to work for now, but the specter of resistance is already lurking. If these drugs are too widely or too improperly used, there is a good chance that resistance mutations will eventually render these drugs ineffective. Universal drug resistance may already be the fate of the drugs amantadine and rimantadine, built on an adamantane backbone (1). The adamantane drugs inhibit the M2 proton channel from influenza A, a tiny tetrameric protein that equalizes pH between the virus and the endosome of the cell that has swallowed it. This process releases the virus contents so that they can do their damage to the cell, so these medicines can significantly retard the infection process. Or rather, they could, if so many influenza strains didn’t harbor the S31N mutation that almost completely nullifies their effect. If we are to develop new drugs to attack the M2 channel, it would be helpful to know how this mutation causes drug resistance. Over the past few years a great deal of structural evidence has accumulated showing how adamantane drugs work on the older, non-resistant channels. The problem is that the evidence supports two different models of M2 inhibition, and so far it has proven difficult to determine which of them is probably correct.

How the question arose

The controversy is the result of two structures published in Nature early in 2008 (2,3). The first of these is a crystal structure of a tetramer of peptides encompassing the transmembrane (TM) region of the M2 channel reported by the DeGrado group at UPenn, which you can see at right (explore this structure at the PDB, noting that the numbering is off by 21). In the detergent used for crystallization, the peptides form a tetramer with a roughly conical pore, which amantadine (purple in these models) physically occludes, giving rise to the pore-blocking model (PBM). This model is consistent with previous results indicating that a single amantadine molecule is sufficient to inhibit the proton channel. In addition, in this model the drug binding site is adjacent to S31 (blue side chain), which is what we’d expect given that an S31N mutation is responsible for most amantadine resistance. The authors propose, given the position of the S31 side chain, that the mutant asparagines form a hydrogen-bonded network that is too constricted for amantadine to bind. Click on the picture for a larger view.

An alternative model was proposed by Schnell and Chou from Harvard University (3). They produced an NMR structure (left) of a 42 amino-acid peptide from M2 encompassing the TM region and an additional C-terminal helix (explore this structure at the PDB). In their structure, taken at pH 7.5 in detergent micelles, the tetramer forms a roughly cylindrical pore that is blocked by the side chains of the known gating residues W41 and H37 (light green in these models). Their structure shows rimantadine bound at four sites near the base of the helix but not in the pore. Using pH-dependent conformational exchange experiments, Schnell and Chou showed that a decrease in pH caused rapid structural changes in the channel, motions that rimantadine slowed. On the basis of this evidence, they proposed a mechanism in which protonation of the gating histidines destabilizes the packing of the TM helices and allows the conductance of protons. Rimantadine blocks the channel by stabilizing the helices, thus this is a dynamic quenching model (DQM). The position of S31 in this model is also somewhat different than the crystal structure, although these models were made at different pH conditions and so this may represent a difference between the closed and open states of the channel.

The distinction here is important. If Stouffer et al. are correct, then drug development should abandon the adamantane backbone altogether and start with a set of significantly different leads to address the resistance problem. The PBM implies that any molecule large enough to occlude the pore will be too large to fit in there following the S31N mutation that induces amantadine resistance. If the DQM is correct, however, then it is conceivable that further refinements to the adamantane base, or similar molecules, could improve affinity enough to overwhelm the mutational effect.

Unfortunately, neither result is unimpeachable. Although it agrees with a great deal of experimental evidence, the low resolution of the crystal structure means that the electron density called amantadine cannot be assigned unambiguously. It is also curious that a hydrophobic molecule like amantadine would bind tightly in the hydrophilic pore. In addition, the crystal form with amantadine bound contains a mutation, G34A (black side chain), which is near the drug binding site and could conceivably have altered the binding specificity of the protein.

The NMR structure has the advantage that it directly includes distance information in the form of NOEs. However, the authors used 40 mM rimantadine to obtain these results, meaning that there were as many rimantadine molecules in the solution as phosphate buffer molecules. Under these conditions, it is possible that the drug bound to a secondary, low-affinity site. Even if this is what happened, it is strange that the rimantadine never bound to the high-affinity site indicated by the crystal structure.

Both experiments use significantly truncated constructs and highly artificial systems to mimic a membrane environment. The structure of any membrane protein depends in often unexpected ways on the composition of the lipid bilayer in which it is embedded and on the structure of that bilayer. The intense curvature of the micelles may have distorted the structure in the NMR experiment, and possibly inappropriate lipids may have had effects on both structures. We know these considerations are relevant for this system, because Schnell and Chou report that the construct used for the crystal structure would not form stable tetramers in the micelle system. Also, as Chris Miller notes in his commentary on these papers (4), there were questions about both constructs with respect to their proton conductivity. Lacking significant stretches of the protein and placed in these environments, it is possible that both structures deviate from in vivo reality in significant ways.

Because the conditions diverge so much, it is difficult to weigh the mechanisms based on these structures alone. The binding site identified by Schnell and Chou is only at the very end of the construct used by Stouffer et al.. In addition, the inhibited crystal structure comes from a low-pH condition while the NMR structure exclusively represents a high-pH condition. Given these differences in conditions, it is not impossible that both models, in whole or in part, are correct. We must turn to additional experiments and alternative evidence to choose between them, specifically data on the stoichiometry of binding and the effects of mutations.

Binding stoichiometry

The crystal structure shows a single binding site for the drug, while the NMR structure implies four, and this is at odds with existing results that indicate that a single molecule of drug is sufficient to inhibit a single channel. Given the homotetrameric nature of the M2 channel, it is in principle not possible for the NMR experiment to distinguish between a single rimantadine binding event and four. That is, the NMR experiment cannot tell us whether the rimantadine-M2 inhibition occurs with a single binding event or requires four drug molecules to bind. Therefore, to argue that DQM is inconsistent with 1:1 stoichiometry overstates the case somewhat.

It may also be somewhat overstating the case to say that there is only one amantadine binding site on M2. Washing amantadine out of your buffer does not reverse inhibition, in part because of slow kinetics of leaving the binding site and in part because these drugs, being very greasy, preferentially partition into the lipid membranes and are therefore not readily removed from a system when its aqueous phase is replaced. It is difficult to measure a binding constant for the drugs because the equilibria under consideration will be quite complex. The studies often cited on the 1:1 stoichiometry (5,6) use structural and kinetic evidence to get at this question.

Czabotar et al. (5) measured tryptophan fluorescence in M2 as a function of pH and rimantadine concentration. They found that fluorescence from W41 was quenched by decreased pH, but recovered when 1 equivalent rimantadine per tetramer was added. This result implies that structural or dynamic changes caused by histidine protonation are reversed by rimantadine inhibition, but this is so general that it cannot be taken to support either the PBM or DQM.

Wang et al. (6) measured the reduction of surface currents in X. laevis oocytes after addition of various concentrations of amantadine. From these results they are able to construct a Hill plot with a coefficient of 1, showing that binding of amantadine is not cooperative. In further results, Wang et al. find that amantadine inhibits M2 channels slightly better at high pH (when the pore is closed) than at low pH, and that amantadine inhibits proton conductance in either direction (rather than favoring one). Both these outcomes are unexpected for PBM, but can be easily explained by DQM. However, the differences in the binding constants are relatively minor and the linearity of the current-voltage relationship may result from some other idiosyncratic feature of the M2 channel, so these results are not unequivocal.

Neither experiment refutes DQM because they do not measure the number of binding sites, but rather the number of efficacious binding sites. If there are four binding sites, but 95% or more of the inhibitory or structural effect is caused by the first drug molecule bound, then these experiments would be unable to distinguish DQM from PBM. Overall, the evidence on the question of binding stoichiometry does not eliminate the possibility of four binding sites existing, but it does place a requirement on DQM that the inhibitory effect of amantadine on the tetramer result from a single binding event. Because the proposed DQM binding site for rimantidine lies between monomers and is linked to the gating tryptophan, this is not unbelievable. Other evidence from these experiments is equivocal, but can be seen as somewhat more problematic for PBM than DQM.

Functional effects of mutations

A serious problem for DQM is that the mutations known to give M2 resistance to adamantane drugs are all located near the PBM binding site. In particular, S31 is adjacent to the drug in the crystal structure and quite distant in the NMR structure. As Miller notes in his commentary, mutational studies are substantially more difficult to interpret than is typically suggested, so this isn’t absolutely probative. In general, however, one predicts mutations to have short-range rather than long-range effects, so at least some resistance mutations ought to evolve at the binding site. However, many of the residues surrounding the DQM site are almost absolutely conserved, presumably because they are essential to the function of the channel. As a result, it would be very difficult to interpret any studies on point mutants in this area. What would be ideal, however, would be to find a set of mutations that produced a functional protein and abrogated amantadine inhibition.

This is the basis for an interesting experiment conducted by the lab of Robert Lamb and reported last year in PNAS (7). In this case, the authors took advantage of the fact that the M2 protein from influenza B virus is not sensitive to adamantane drugs. They constructed a chimeric protein containing about a dozen residues from influenza A M2 — specifically, the dozen or so residues surrounding the PBM site. If PBM is correct, then we would expect that these residues, which define that site, would impart amantadine susceptibility to the influenza B channel. This is what happens, sort of. For your benefit, I have shamelessly stolen their figure (right), but you can check out this paper yourself because it is open access. In this assay, again involving X. laevis oocytes, the hybrid channel is sensitive to amantadine (bottom trace), but only half as sensitive as the wild-type influenza A channel (second from top). This result suggests that there is important context conferring susceptibility outside the PBM site. However, this could be something as simple as helix orientation, so the result does not necessarily imply that there is an external binding site.

Additionally, the authors made point mutations at residues (L38, D44, and R45) that were presumed to be important in the DQM mechanism or have long-range effects on amantadine binding. None of these mutations appeared to affect amantadine resistance. In contrast, experiments in liposomes reported by the Chou group this May showed that a D44A mutation prevented rimantidine from having an effect (8). This conflict in results is difficult to reconcile, but may result from the different constructs used (the Chou group used a truncated form of M2 while the Lamb group used the full-length protein) or from changes in ion specificity caused by the D44A mutation. It might be of value to repeat these experiments with the alternative construct: truncated in oocytes, full-length in liposomes. Because the D44A mutant does not appear to conduct protons as efficiently as WT, the proposition that the function of this mutant is too deranged to provide trustworthy information should also be considered.

Additional experiments in the Chou paper are meant to address the relationship between the DQM site and the mutations at the PBM site. They show that the S31N mutation prevents rimantidine binding to the remote site, and also that this mutation makes the protein generally more dynamic. From this evidence they propose that this mutation, at least, disrupts amantadine binding by destabilizing the helical packing of the channel and thus interfering with the organization of the lipid-facing pocket.

They also examine an S31A mutation and find that it is not rimantadine-resistant or destabilizing to the packing. This supports their dynamic model in a limited way, because it demonstrates that only certain mutations at the S31 site will generate resistance. It does not cast any doubt on PBM, however, because in that model resistance in the S31N mutant is explained by the idea that its side chain will partially obstruct the pore so that rimantadine will not fit. I do not think it was ever proposed that specific contacts between S31 and the drug stabilize the binding; in fact, the general absence of such contacts strikes me as a concern about PBM.

Chou et al. also examine the effect of rimantadine on the shorter construct used for the crystal studies. They find that the inhibition of this construct is substantially weaker. However, it also conducts protons at a much slower rate in this assay, suggesting that there may be additional serious problems with the function of this construct. It may be that it simply is not appropriate to use this construct for studies in solution or living membranes. That doesn’t necessarily imply that this peptide will give incorrect information in the stabilizing environment of a crystal.

What do we know, and what do we need?

Very little of this evidence unequivocally prefers one model to the other. We know that a single adamantane molecule is sufficient to inhibit M2, and while this is most obviously compatible with PBM it need not be inconsistent with DQM. It is also apparent that various constructs of the M2 channel retain adamantane susceptibility after ablation of the DQM site, either by truncation, mutation, or the construction of a chimeric protein. In all assays, however, the adamantane drugs lose a considerable amount of inhibitory power, so these results are not entirely consistent with PBM either. And, at least in the Chou lab’s assays, interference with the DQM site also reduces adamantane susceptibility and deranges function. Moreover, the NMR data from the Chou lab shows that mutations at PBM site have a long range effect on the DQM site, which mitigates the probative power of the S31N mutation.

How do we address this question? One important step would be to start comparing like to like. We are considering evidence from a plethora of constructs and conditions, and the evidence in conflict is often collected in very divergent experiments. Ideally we would like to have structures of the wild-type channel at low and high pH in a lipid environment that closely mimics the composition and curvature of a mature influenza virion. As this is unlikely in the near term, we must hope for NMR and crystal structures that at least use the same construct, minimally mutated, under similar conditions. NMR studies at low pH would be of value in assessing whether these studies in fact contradict one another. Additionally, it would be useful to make adamantane derivatives labeled with a free radical or other paramagnetic label; this would presumably allow the identification of a binding site at lower drug concentrations in an NMR experiment. Labeling the drug with a metal might also allow its identification in a crystal structure without any need to push the resolution significantly higher. Finally, actual structures of the S31N mutant, positively identifying the disposition of this side chain, would be of great value in judging the question.

Structural experiments can take a great deal of time and careful tuning, a requirement exacerbated by the often-fickle behavior of membrane proteins. As such, additional mutational studies could prove useful. Inverting the chimera experiment of Jing et al. to create a chimeric protein with the upper channel from influenza B and the lower channel from influenza A may be a helpful supplement to the existing experiments. If the C-terminal portion of the channel makes a contribution to adamantane inhibition this chimera will also be rimantidine-sensitive. In addition, new mutations at S31 could help distinguish the possibilities. The PBM supposes that the N31 side chains stick into the pore and form hydrogen bonds, while the DQM supposes that they stick into the interface of the TM helices and destabilize them. An S31L mutant should disrupt the helical packing but not form hydrogen bonds or extend the L31 side chains into the pore. If functional, such a mutant ought to be rimantidine resistant if the DQM is correct, but not if PBM is correct. Assuming the geometry of the longer side chain is wrong for formation of a hydrogen bonding network, an S31Q mutation might be useful as well. Similarly, mutations that increase the size of the L40, I42, or L43 side chains could prevent adamantane binding to the DQM site without degrading the channel’s transport capabilities; the drug sensitivity of such a mutant would be a powerful argument either way. Any experiments of this kind would likely be easier to perform than to interpret, but could provide valuable insight. Obviously, it would also be important to establish that each mutant was competent at transporting protons.

The unspoken assumption of the debate so far is that these mechanisms are mutually exclusive, but there is no particular reason to believe this must be so. The structural experiments definitively show that binding to both sites is at least possible — even if one clings tenaciously to the idea that the density observed in the crystal is not in fact amantadine, that structure at least shows that the PBM site is capable of accommodating the drug. It might therefore be plausible that adamantane drugs inhibit M2 using both mechanisms simultaneously, or that DQM predominates at high pH and PBM at low pH. Redundancy in inhibitory mechanisms may explain the curious features of amantadine inhibition noted by Wang et al., and the inability of experiments specific to a single site to completely account for adamantane inhibition. In addition, the fact that S31N interferes with both mechanisms may explain why it is the primary resistance mutation.

An experiment with the alternate chimera mentioned above could test this possibility. In addition, if the mechanisms switch off in a pH-dependent fashion, then this should be testable with the hybrids: specifically, the A/B M2 used by Jing et al. should have lower susceptibility to adamantane drugs at high pH than at low pH. Similarly, the B/A M2 chimeric protein, if inhibited by amantadine, would be more resistant at low pH.

Doubtless these suggestions are nothing new to the members of the labs working on this perhaps unexpectedly hairy question. Membrane protein structure and function is one of the most difficult experimental subjects in biochemistry, and constitutes a critically important frontier in scientific efforts to improve human health. It is infinitely easier to propose most of these experiments than it is to perform them, and I would be remiss if I did not temper the persistently critical tone of this post with some praise for the efforts of all the scientists involved in this research, and for their commitment to getting the right answer. These papers represent years of work by incredibly talented people using some of mankind’s most advanced scientific techniques. The lack of clarity on the question of adamantane drugs binding to M2, even in the face of this amazing effort, is a testament to the enormous difficulty of researching these critical systems.


1. Deyde, V., Xu, X., Bright, R., Shaw, M., Smith, C., Zhang, Y., Shu, Y., Gubareva, L., Cox, N., & Klimov, A. (2007). Surveillance of Resistance to Adamantanes among Influenza A(H3N2) and A(H1N1) Viruses Isolated Worldwide The Journal of Infectious Diseases, 196 (2), 249-257 DOI: 10.1086/518936 OPEN ACCESS

2. Stouffer, A., Acharya, R., Salom, D., Levine, A., Di Costanzo, L., Soto, C., Tereshko, V., Nanda, V., Stayrook, S., & DeGrado, W. (2008). Structural basis for the function and inhibition of an influenza virus proton channel Nature, 451 (7178), 596-599 DOI: 10.1038/nature06528

3. Schnell, J., & Chou, J. (2008). Structure and mechanism of the M2 proton channel of influenza A virus Nature, 451 (7178), 591-595 DOI: 10.1038/nature06531

4. Miller, C. (2008). Ion channels: Coughing up flu’s proton channels Nature, 451 (7178), 532-533 DOI: 10.1038/451532a

5. Czabotar, P., Martin, S.R., & Hay, A.J. (2004). Studies of structural changes in the M2 proton channel of influenza A virus by tryptophan fluorescence Virus Research, 99 (1), 57-61 DOI: 10.1016/j.virusres.2003.10.004

6. Wang, C., Takeuchi, K., Pinto, L.H., & Lamb, R. (1993) Ion Channel Activity of the Influenza A Virus M2 Protein: Characterization of the Amantadine Block J. Virol. 67 (9) 5585-5594 Available free from PubMed Central

7. Jing, X., Ma, C., Ohigashi, Y., Oliveira, F., Jardetzky, T., Pinto, L., & Lamb, R. (2008). Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel Proceedings of the National Academy of Sciences of the United States of America, 105 (31), 10967-10972 DOI: 10.1073/pnas.0804958105 OPEN ACCESS

8. Pielak, R., Schnell, J., & Chou, J. (2009). Mechanism of drug inhibition and drug resistance of influenza A M2 channel Proceedings of the National Academy of Sciences of the United States of America, 106 (18), 7379-7384 DOI: 10.1073/pnas.0902548106

The Scientific Activist and Discovering Biology in a Digital World also have interesting posts on this subject.

Jan 072009
 
ResearchBlogging.orgAntibiotics such as chloramphenicol suppress infections by inhibiting bacteria from making proteins. They achieve this by binding to and blocking the peptidyl transferase center (PTC) of the ribosome, a large complex of RNA and protein that performs nearly polypeptide synthesis in living cells. Although PTC-binding antibiotics comprise several different families of compounds, mutations in the ribosome that confer resistance to one family often produce cross-resistance to other families. This is difficult to understand because the PTC itself is highly conserved and not very tolerant of mutations. In an upcoming paper (open access, read along) in the Proceedings of the National Academy of Sciences, a team of researchers from the Weizman Institute of Science analyze several crystal structures of the ribosome to understand how this cross-resistance arises.

Davidovich et al. mapped nucleotide mutations known to confer resistance to PTC antibiotics onto x-ray crystal structures of the large ribosomal subunit from D. radiodurans in complex with antibiotics. One interesting facet of the resistance mutations became immediately apparent: they were almost all clustered on one side of the antibiotic binding site.

You can see this pretty clearly in Figure 2 panels B&D. Although the antibiotics (large pink surface) are surrounded by nucleotides, most of those that are on the left side (thin tan sticks) do not confer resistance if mutated. Resistance-conferring mutations instead cluster around the “rear wall” of the PTC (to the right). The authors explain that in this region ribosomal functions primarily rely on the sugar-phosphate backbone of the rRNA. Because the backbone elements are the same for all ribonucleotide bases, mutations in this region are more likely to be tolerated without significant loss of function.

Another striking feature of resistance mutations is visible in Figure 2 and quantified in Figure 3A, namely that many of these mutated bases do not contact the antibiotics directly. In particular, mutation of G2032 appears to play a role in conferring resistance to several different antibiotics. Overall, however, it appears that numerous long-range interactions can interfere with antibiotic binding.

The lynchpin of these interactions seems to be U2504, a base that directly contacts the bound antibiotic in most cases. Mutations to U2504 itself do not appear to be well-tolerated, but many of the long-range mutations occur in the layer of bases surrounding it. The authors describe in detail several mechanisms by which the observed mutations might increase the flexibility of U2504, allowing it to adopt positions that could allow continued protein synthesis while reducing the binding of antibiotics. The commonality of interactions with U2504, and the importance of the structural context of the surrounding nucleotides, explains why many mutations can give rise to cross-resistance.

The practical upshot of these findings is that they may serve as a guide for the design of future antibiotics. Since the majority of the drug-resistance mutations lie on the rear wall of the PTC, the effectiveness of these antibiotics may be enhanced by improving their binding to other parts of the site. With further modeling it may also be possible to design antibiotics that can compensate for flexibility at U2504. These findings also remind us that dynamics and long-range interactions can be important to the function of any biomolecule with a folded three-dimensional structure, not just proteins.

C. Davidovich, A. Bashan, A. Yonath (2008). Structural basis for cross-resistance to ribosomal PTC antibiotics Proceedings of the National Academy of Sciences, 105 (52), 20665-20670 DOI: 10.1073/pnas.0810826105 OPEN ACCESS

 Posted by at 4:00 AM
Sep 192008
 
ResearchBlogging.orgBiochemists often rave about the great wonders of enzymes, lavishing praise on the prodigious rate enhancements they produce, and their exquisite positioning of functional groups. One can quite reasonably ask how such magnificently useful proteins came into being. One accurate answer, of course, is that after a couple hundred million years evolution can get almost anything right. Another answer is that most enzymes come from other proteins, via a process called gene duplication. The genetic changes that follow one of these duplications turn two copies of one protein into two completely different proteins with diverse activities.

Gene duplication events are infrequent errors of DNA replication or repair. Diploid eukaryotes such as ourselves carry two copies (or near-copies) of most genes as a matter of course, but gene duplications produce extra copies beyond that. In theory, the presence of these extra copies of a gene means that one of them can mutate freely, without the pressure of carrying out its normal job. When it drifts into a useful function, selective pressure is again applied, causing a refinement of the active site to maximize the efficiency of the new activity. The overall scheme looks something like this:

Duplication → Divergence → Refinement

It may seem incredible that a vast diversity of protein structures and activities can arise simply by making copies, even imperfect copies. However, certain quirks of the translation machinery mean that small changes in DNA can amount to enormous changes in a protein’s topology. For instance, an insertion or deletion of a single base can cause a frameshift mutation, producing a protein that bears no resemblance to its progenitor despite having only 1 different base pair. Many DNA triplets that normally encode amino acids are only a single base-pair mutation away from becoming a stop codon, truncating a protein and likely changing its structure significantly. Similarly, stop codons can be easily eliminated, producing much larger proteins. In eukaryotes, point mutations near the borders between introns and exons can cause new regions of DNA to be translated into protein. Of course, drastic changes like these mostly just produce useless junk, but occasionally a novel fold or function arises.

More conservative alterations of a gene sequence can still produce significant changes. As I’ve mentioned before on this blog, some members of the Cro family of proteins have very high sequence identity and yet possess different structures. I also have not yet tired of reminding you that the chemokine lymphotactin has two different structures with a single sequence, either of which can be stabilized into an exclusive fold by a point mutation.

Additionally, research from the lab of John Orban shows that a mere 7 mutations are required to convert the engineered protein GA88 (PDB) into a completely different structure, GB88 (PDB) (1). These proteins were previously shown to have different folds and functions, but the contrast between the high resolution structures (shamelessly stolen figure on the right) is striking. Moreover, the Orban lab has refined this system so that the structural conversion can be effected with only three mutations, rather than seven. What all this research indicates is that the transitions that convert a sequence from one fold into another may be sharper than previously realized; even a relatively small number of fairly conservative mutations may be able to completely transform a protein’s structure.

For all that, most new enzymes arising via gene duplication resemble their ancestors in identifiable ways. Often the two proteins perform the same chemical steps, and the novel function amounts to a different substrate specificity. This suggests the possibility of an alternate mechanism of gene duplication, in that a protein could evolve a novel specificity while retaining its original function. Diversifying its activities in this way would probably limit an enzyme’s catalytic effect in both reactions, but a subsequent gene duplication event would allow each copy to refine its particular reaction. The scheme would look like this:

Diversification → Duplication → Refinement

The advantage of this model, from an adaptationist’s perspective, is that it brings selective pressure to bear at every step. Once a new function has evolved in response to environmental conditions, duplicating the gene may provide an organism a concrete advantage. After duplication, the advantage of separately refining the two activities is obvious.

The two models are not as different as they might seem at first glance, because nearly every enzyme catalyzes two reactions anyway, that is, the forward and reverse reactions of an equilibrium. A “new” activity for a given enzyme can therefore result from something as simple as being targeted to a different cellular compartment or a change in specificity that involves an oppositely-oriented equilibrium.

The most obvious objection to the latter model is that during the period of gene sharing prior to duplication, neither protein function will be very efficient. As a matter of fact, the appearance of a new activity does not always impair an enzyme’s ability to do its original job (and indeed can even enhance that activity). Still, because of the exquisite tuning of enzyme active sites we can expect that many modifications to this region will reduce catalytic power. That being the case, how might an organism survive or thrive during the gene-sharing period? The answer, which always seems obvious in retrospect, is to make more of the less efficient enzyme, as was demonstrated in a recent paper by Sean Yu McLoughlin and Shelley Copley (2).

McLoughlin and Copley took a strain of E. coli that lacked an enzyme, ArgC, that is critical for glucose metabolism. They treated these bacteria with a strong mutagen and then picked a colony that grew well on uncomplemented glucose. After showing that these bacteria had developed a novel activity equivalent to ArgC, they isolated the “new” enzyme and found that it was actually an existing enzyme, ProA, which performs similar chemistry. This enzyme had gained the ability to take over the tasks of the missing ArgC, enhancing the rate of that reaction 12-fold. The actual chemistry of these reactions was quite similar, but in gaining the ability to operate on ArgC’s substrate, the activity of ProA towards its own substrate was reduced 2800-fold. The bacteria compensated for this by upregulating the production of the enzyme. A second mutation in the promoter region of the gene was helpful, but not necessary, in this respect.

Because enzymes are catalysts, a small increase in protein concentration can result in a significant increase in the availability of the reaction products. Biochemists often say, seeing a 3000-fold reduction in activity, that an enzyme is dead. The reality is that it’s just slower, and a living thing can compensate for that in ways not available to an isolated reaction in a test tube. Organisms have shown that they have ways to survive what an enzymologist might see as fatal.

Of course, modern bacteria benefit from a number of well-tuned regulatory and feedback mechanisms that allow them to sense when particular metabolites are running low and to increase the production of proteins that can replenish them. Earlier, more primitive organisms might not have had these expedients available. Could they have survived gene sharing?

Too little is known about early life forms to answer such a question definitively. However, it is interesting to note that one method of making more protein is to make more of the gene. That is, the concentration of a deficient enzyme can be increased via gene duplication. By a fortuitous coincidence, a single mechanism could both enable an organism to tolerate reduced enzymatic efficiency and allow the evolutionary process to independently refine its activities.

It is also worth bearing in mind that just as ancient organisms did not necessarily resemble modern ones, ancient proteins might not have resembled the modern item. The exquisite positioning of functional groups that characterizes modern enzymes requires a rigid fold and contributes significantly to the rate accelerations they produce. However, substantial rate enhancements can still be achieved in the absence of a stiff native state.

One occasional result of mutations is the formation of a molten globule, a protein that lacks a stable fold but still exists in a collapsed state with something resembling a hydrophobic core. Although that doesn’t sound particularly useful, many molten globules have enzymatic or other functional activities. Recent computational studies on a molten-globule mutant of Methanococcus jannaschii chorismate mutase suggest that realistically low energy barriers can be achieved by a broader array of structural states in these proteins (3).

Researchers from the lab of Arieh Warshel used a simplified model to sample the conformational space available to the molten globule enzyme (mMjCM) and a stably folded form of the enzyme (EcCM). As you might expect, the lowest-energy conformations are much more diverse for mMjCM than for EcCM. Roca et al. then computed the energy barrier for catalysis for conformations that closely resembled the ideal structure (region I), conformations which had most of the groups in the right general position but were significantly removed from the ideal (region II), and conformations that did not resemble the ideal at all (region III). For EcCM, only structures in region I had energy barriers low enough to plausibly allow catalysis. The molten globule, however, had energy barriers that would allow catalysis in region I and region II. You can see this in the figure below, which I shamelessly stole from their paper: the dotted orange line corresponds to a 16 kcal/mol energy barrier, what they felt to be the largest barrier reasonable for a catalyst. The results for mMjCM are on the left, EcCM on the right.

The upshot of this is that molten globules may be able to maintain catalytic power in the face of structural diversity that causes folded proteins to fail. While the stable fold produces greater rate enhancements (note that EcCM has lower energy barriers), the molten globule tolerates a wider array of structural conditions. Consequently, proteins of this kind may be much more amenable to the addition of new functions. So long as an appropriate orientation of functional groups is reasonably likely, a protein without a rigid conformation can still achieve impressive rate enhancements.

Conceivably, an early molten globule enzyme could have the ability to catalyze several different reactions, switching between the required conformations as needed, without a significant loss of catalytic power to any of them. Duplication of a multi-functional molten globule like this would allow each chemical function to be refined independently, with additional duplications and refinements giving rise to substrate specificity.

The different models of gene duplication each have their own explanatory advantages, and the available evidence suggests that new proteins and enzymatic activities have evolved (even within the last century) using both routes. As this is one of nature’s favored methods of generating novel activities, so it is becoming ours. The artificial enzymes recently produced by David Baker’s lab were designed onto an existing protein scaffold in what could be taken as a computational mimicry of the gene duplication process.

1. Y. He, Y. Chen, P. Alexander, P. N. Bryan, J. Orban (2008). NMR structures of two designed proteins with high sequence identity but different fold and function Proceedings of the National Academy of Sciences, 105 (38), 14412-14417 DOI: 10.1073/pnas.0805857105

2. S. Y. McLoughlin, S. D. Copley (2008). A compromise required by gene sharing enables survival: Implications for evolution of new enzyme activities Proceedings of the National Academy of Sciences, 105 (36), 13497-13502 DOI: 10.1073/pnas.0804804105

3. M. Roca, B. Messer, D. Hilvert, A. Warshel (2008). On the relationship between folding and chemical landscapes in enzyme catalysis Proceedings of the National Academy of Sciences, 105 (37), 13877-13882 DOI: 10.1073/pnas.0803405105

 Posted by at 1:00 AM
Aug 302008
 
ResearchBlogging.orgIt is rare, but not unheard of, for a human baby to be born with a tail. Atavism of this kind is generally understood to be the result of mutations in regulatory genes that cause an ancestral pattern of development to re-emerge. A physiological step backwards through the path of descent is often easy to recognize, because many of the evolutionary relationships are known. It should also be possible to identify atavistic events in particular molecules. For instance, one can imagine that a mutation to CLC-0 might result in a reversion to the ancestral transporter function. In a recent article in PLoS Biology, researchers from Florida State University and Brandeis University identify just such a relationship in the bi-functional enzyme inosine monophosphate dehydrogenase (IMPDH). PLoS Biology is an open-access journal, so open it up and follow along.

IMPDH plays a critical role in the synthesis of guanine nucleotides, an essential component of DNA. Two reactions take place in the active site — first, the inosine ring is oxidized to xanthosine, forming a covalent linkage with the enzyme, and then this bond is broken by a hydrolysis. The enzyme active site changes shape to carry out the reaction, bringing a catalytic arginine (R418) into position to activate the water for nucleophilic attack. Any time you see a complicated mechanism like this, it’s natural to wonder how such a system could have evolved. Min et al. performed simulations and experiments to find out.

Using a crystal structure of IMPDH as a starting point, Min et al. performed hybrid QM/MM simulations in which the atoms taking direct part in the reaction were treated with quantum mechanics, and the rest of the protein was simulated using molecular mechanics. As one would expect given the enormous reduction in catalytic rate that occurs when R418 is mutated, the reaction proceeded through the arginine when the simulation had a neutral R418 side chain. The water is stabilized by two additional side chains from T321 and Y419, and reacts almost instantaneously, without the formation of a stable hydroxide intermediate. Although this is unusual, this prediction of the simulation is consistent with isotope effect experiments.

When the arginine was replaced by a glutamine in the simulation, the mechanism changed, naturally. Under these conditions, it was Y419 that activated the water for the hydrolysis, although the energy barrier was much higher (leading to a slower reaction). Again, the characteristics of the reaction indicated by the simulation line up pretty well with the results of biochemical experiments. Of course, Y419 enters the active site the same way R418 does, so the question of how the hydrolase activity could have evolved remains open.

Something very interesting, however, happens when the simulation is performed with R418 in a charged state. A fully protonated arginine will have a very hard time activating water for a nucleophilic attack. The simulation indicated that under these conditions, T321 performed this role, after being activated by a nearby glutamate (E431). T321 is adjacent to cysteine 319, which is essential for the oxidation reaction, and is not located on the mobile flap. If T321 really can catalyze hydrolysis, this would mean that it is possible that IMPDH possessed an (inefficient) hydrolysis activity before it evolved the mobile flap.

Because T321 only plays a signficant role in catalysis when R418 is protonated, blocking this pathway should result in decreased IMPDH activity at low pH. This is precisely what Min et al. observe in enzymatic assays (Figure 5) on a mutant in which E431 is mutated to glutamine. There is other experimental support as well: IMPDH enzymes that have been mutated at R418 usually have large isotope effects, which makes sense in light of the fact that the alternative T321 pathway involves the simultaneous transfer of two protons (rather than just one).

Things get even more interesting when IMPDH is compared to one of its cousins, GMP reductase. Although GMPR catalyzes a very different reaction, the C319/T321/E431 triad is also present there. This, along with other data from sequence alignment, suggests that these three residues were also present in a similar configuration in the ancestor of these modern proteins. Over time, progressive optimization of the two proteins resulted in the T321 pathway being supplanted by the more effective R418 in IMPDH, while remaining essential in GMPR.

If T321 really is a remnant of an earlier water-activating pathway, why is it conserved now that IMPDH has a much more efficient catalytic residue available? T321 is probably preserved because it stabilizes the water while it is being activated by R418. However, the other essential residue of that activating pathway (E431) is usually an inactive glutamine in eukaryotic forms of IMPDH (and some prokaryotes, as well). In these species the T321 activation pathway has been completely supplanted by the arginine pathway. Yet in the other forms of IMPDH this alternative mechanism still lingers, perhaps because of the additional activity it affords at low pH, or because it confers resistance to a particular inhibitor of the enzyme. In that sense, IMPDH’s “tail” might provide an adaptive advantage quite different from that which gave rise to hydrolytic activity in the first place.

Donghong Min, Helen R. Josephine, Hongzhi Li, Clemens Lakner, Iain S. MacPherson, Gavin J. P. Naylor, David Swofford, Lizbeth Hedstrom, Wei Yang, Daniel Herschlag (2008). An Enzymatic Atavist Revealed in Dual Pathways for Water Activation PLoS Biology, 6 (8) DOI: 10.1371/journal.pbio.0060206 OPEN ACCESS
Disclaimer: Although I have little contact with Dr. Hedstrom’s group, I am also working at Brandeis.

 Posted by at 7:30 PM