Sep 192011

Given that videogames are often demonized by research (and “research”) blaming them for everything from rudeness to the epidemic of youth violence, gamers often take a great deal of cheer from research attaching positive outcomes to videogame play. One such article that recently attracted some attention was work suggesting that playing videogames could correct amblyopia (often called “lazy eye”) in adults (1). Of course, given how negative results get oversold, it’s worth asking whether these have been, too. The paper appeared in the open-access journal PLoS Biology, so let’s open it up and take a look.

The fundamental problem that the authors are out to solve is that, while amblyopia can generally be corrected if it is treated in childhood, success tends to be rarer in adults. Knowing that video games have proven useful in improving adults’ abilities to perform a wide variety of visual tasks, these researchers decided to ask whether they could help treat amblyopia.

Figure 1 shows their experimental design. First they screened and assessed a group of adults with amblyopia. Then they divided these individuals into three groups. One group (10 individuals) played a total of 40 hours of Medal of Honor: Pacific Assault with the normal eye patched. An additional 3 individuals were assigned to a group that played SimCity Societies for an equal amount of time (it is unknown whether the author’s controlled for Societies‘ well-known liberal bias), again with the normal eye patched. The final seven individuals were given twenty hours of ordinary visual challenges (watching movies, reading, etc.) with the normal eye patched (occlusion therapy or OT), in order to ensure that patching alone wasn’t causing any observed improvements. Most individuals from the last two groups, following an intermediate assessment, then went on to play 40 hours of MOH.

As the authors note, there are several limitations to this study immediately apparent. The sample size was small, individuals were not assigned to groups randomly, and both participants and researchers knew what kind of treatment they were getting. This does not mean we should disregard the results. However, they do need to be taken with a grain of salt until the findings can be replicated in a larger sample.

And there is good cause to try to replicate these findings. Figure 2 is, unfortunately, something of a symbol party (the symbols and colors identify individual subjects by their type of amblyopia), so we’re better off focusing only on panel D, at lower right. The first item in panel D is a logMAR chart, used to measure visual acuity, and it probably looks familiar to you. Each line on the chart represents 0.1 logMAR units, and as you can see, the lower the score, the better your vision. The panel to the right of that shows the averaged data from all twenty individuals after OT and videogame therapy (VG). Here they are showing the percentage improvement in acuity in crowded conditions (the whole chart) or in isolation (a single letter). OT did not produce any improvement in acuity, while 40 hours of VG therapy produced an average 30% improvement in acuity. The other two graphs here indicate that improvement in acuity was unrelated to baseline acuity, and that the crowding index (the loss of acuity due to the presence of other letters) did not change substantially due to therapy.

This is a critical figure because, as the authors state, “reduced visual acuity is the sine qua non of amblyopia.” Substantial improvements in acuity, therefore, represent a major goal of therapy. Perceptual learning, in which participants make subtle visual judgments using their amblyopic eye, has been shown to improve acuity in adult amblyopes as well. If videogames can produce a comparable improvement, however, they may prove just as efficacious because they encourage therapy (=play) through fun.

Panels A-C of Figure 2 show the raw results and percentage improvements for each individual group. Two additional points are worth noting. Panel B shows that the 20h of occlusion therapy were ineffective, but the subsequent 40h of MOH improved acuity in all continuing individuals. However, it should be noted that while the gaming took place at the research location, the occlusion therapy was done on the individual’s own time and self-reported. This study therefore does not control for the benefits of a monitored and enforced eye-exercise regimen.

Panel C is also of interest. Although the group here is small (and the data correspondingly noisy), it appears that their acuity was improved by both SimCity and MOH. This was somewhat unexpected, because in the past positive visual effects produced by action video games have not been replicated by non-action games. Understanding why that’s not the case here may help provide some additional insights into the mechanisms by which games improve acuity in these patients. I haven’t played SimCity Societies, but having played previous SimCity iterations I know that these games often require the player to integrate a variety of visual information (traffic flow, electricity, dynamic economies) simultaneously, which may underlie the observation. Had these subjects actually played videogame chess, their improvement might have been less.

The authors went on to test the subjects’ vision in various ways. Figure 3 shows a test of positional acuity, and is rather badly made, but gets the point across that positional acuity (assessed using the funky little chart in panel A) improved in the game-playing group (panel B). This included both increases in “sampling efficiency”, related to a fitted number of correct positions extracted (out of 8) (panel C and E-SB2) and decreases in “internal noise”, or the degree to which the individual’s own eyes interfere with his assessment of position (panel D and E – SA5). The results in panel E compare improvements in efficiency and internal noise, with the three labeled graphs comparing results in the non-amblyopic eye (NAE) to the amblyopic eye (AE) before and after videogame treatment.

The authors also decided to test the effect of the games on spatial attention, as they report in Figure 4, by briefly showing the subjects a field of dots (at a size where they could be easily seen), followed by a checkerboard pattern and asking them to report the number of dots seen (panel A). Not all the individuals had an appreciable difference between the non-amblyopic eye and the amblyopic eye prior to the VG treatment (panel B). However, the degree of improvement in spatial attention tended to be greater the worse the initial condition was (panel C), including for SimCity players (symbols surrounded by dotted circles). For the worst-off subjects (dotted circle in panel B), significant improvements in accuracy and response time were observed (panel D-F).

Finally, the authors tested the stereovision of some subjects using a standard test (Figure 5). Again, substantial improvements were noted in all those tested (which excluded subjects with strabismus), to the degree that some of them were effectively cured.

These results show that playing video games produced dramatic improvements in vision for adults with amblyopia by a variety of measures. However, this study had many limitations, and nobody should go around prescribing (or self-prescribing) videogames as amblyopia therapy just yet. The sample size here was very small, and because of the way groups were assigned the various populations differed in non-trivial ways (the MOH group, for instance, was younger and more male than the others). The conditions for occlusion therapy were very different from those used in the videogame therapy, which could have contributed to the different outcomes. Even if a more comprehensive trial shows similar results, more work will be necessary to identify the best course of treatment, which I note is unlikely to take the form of a 24-hour Modern Warfare 3 binge fueled by Bawls and pizza.

That said, these results appear to justify a larger, more complete study, which we can certainly hope to see in a few years from these authors.

1) Li, R., Ngo, C., Nguyen, J., & Levi, D. (2011). Video-Game Play Induces Plasticity in the Visual System of Adults with Amblyopia PLoS Biology, 9 (8) DOI: 10.1371/journal.pbio.1001135

Apr 122011

ResearchBlogging.orgThe classic neuropathological hallmarks of Alzheimer’s disease are the appearance of amyloid plaques composed primarily of amyloid beta (Aβ) peptides, and neurofibrillary tangles composed mainly of hyperphosphorylated tau protein. For many years, research into treatments for Alzheimer’s disease proceeded on the hypothesis that the plaques were toxic to the surrounding neurons. More recently, however, evidence has shown that soluble Aβ oligomers may be the primary toxic species. A recent paper in Proceedings of the National Academy of Sciences supports this hypothesis by showing that Aβ oligomers isolated from the brains of Alzheimer’s sufferers cause neuronal degradation and improper phosphorylation of tau (1). This paper is open access, so open it upand read along.

Jin et al. isolated dimers of Aβ from homogenates of human brains from Alzheimer’s patients. Dimers were separated from monomers and higher-order oligomers by size-exclusion chromatography in the presence of a strong detergent that typically breaks up folded proteins and repeating aggregates. This separates the dimers and higher oligomers from each other, and also dissociated weakly-interacting peptides (due to the effects of the detergent). As you can see from Fig. 1A, this produced fractions that contained either detergent-stable Aβ dimers (AD-TBS) or normal cortical proteins (cont-TBS) in identical solution conditions. They also created synthetic dimers by mutating Aβ to contain a cysteine that could form a covalent linkage between peptides (Aβ40S26C). They then used these various materials to treat primary cultures of neurons (that is, neurons that were obtained by directly harvesting them from an animal), with the dimers reaching a final concentration 0.5 nM in the growth medium.

Fig 1B establishes that, among the materials studied here, Aβ dimers are uniquely responsible for the appearance of tau “beads” along the neurites of the cultured cells after 3 days (the widespread dots in the final column of images). This effect is quantified in 1C, which shows that the dimer-containing fractions produced a dramatic increase in this clumping. According to the authors, these easily-visible clumps are only one symptom of widespread problems with the cells’ cytoskeletons. This sort of cytoskeletal trouble is expected because tau’s function is to stabilize and assist in the formation of microtubules from tubulin. The upshot of this figure is that continuous exposure to Aβ dimers (Fig 1D establishes that the dimers persist through the treatment period) appears to cause some sort of trouble with tau, which may reflect the incipient formation of the famous tangles.

The natural follow-up question is whether tau is necessary for this cytoskeletal derangement. The fact that the cultured neurons must mature, with a correlated increase in tau expression, for Aβ dimers to have an effect suggests that it must be. To check this, the authors used RNA inhibition to knock down tau levels. Fig 2A demonstrates that tau, but not tubulin, expression was altered using the tau-specific RNAi (but not the scrambled cont-RNAi). The cytoskeletal damage caused by both the natural dimer and the Aβ40S26C synthetic dimer were suppressed by tau-RNAi (Fig. 2B). At least at this timescale, it therefore appears that normal tau expression levels are necessary for this toxic effect of Aβ dimers. However, as tau in neurofibrillary tangles never breaks down, it seems like a longer exposure to Aβ under these conditions should produce similar toxic effects eventually.

The complementary experiment, is shown in Fig. 3, using a hybrid construct where human tau was fused to a fluorescent protein. As you can see from these images, under control conditions (columns labeled EGFP), cells treated with Aβ monomers and dimers have only subtle differences after two days, and beading is only evident after three days of treatment. When tau is overexpressed (columns labeled tau-EYFP), the cytoskeletal issues are obvious a day earlier. The tau-EYFP appears to be distributed in the same places as normal tau (fourth row), so the EYFP tag probably isn’t responsible for this effect, and the normal behavior of monomer-treated cells is reassuring. However, the EYFP tag may make tau more susceptible to some kind of dysregulation. Because this experiment both increases the total amount of tau and introduces the human protein, the reason for the enhanced susceptibility is difficult to determine. A control experiment in which rat tau-EYFP was expressed in the same construct would have been very helpful in clarifying this point.

As I mentioned above the formation of the tangles is associated with tau becoming highly phosphorylated. Jin et al. therefore made an effort to confirm that this was happening in their cultured cells, using antibodies that would recognize some specific sites in the tau protein that receive phosphate tags. Fig. 4 summarizes the results, indicating that human tau expressed in rat neurons becomes highly phosphorylated at serines 202, 205, and 262. For some reason, the endogenous rat tau did not become significantly phosphorylated at S262; this may have something to do with the apparently enhanced toxicity of Aβ dimers in the presence of human tau.

The paper’s final figure tests whether antibodies directed against specific sites in Aβ can prevent the observed cytoskeletal degradation. They found that two antibodies that bound to the N-terminus of Aβ significantly suppressed the effect of the dimers over the three-day timespan (fourth and fifth columns of A). However, an antibody directed towards the C-terminus of Aβ42 did not have much effect. Fig. 5C suggests that this is because this antibody simply didn’t bind to much of the Aβ in solution, either because most of the isoforms are shorter or because the C-terminus is protected in some way.

These results clearly link cytoskeletal disruptions caused by tau to the presence of soluble Aβ dimers, linking the two well-known pathological hallmarks of Alzheimer’s disease. That soluble oligomers, rather than fibrils, were responsible for the effect does not necessarily prove that the plaques aren’t important, for two reasons. The first is that, while these results clearly demonstrate dysregulation and aggregation of tau, true neurofibrillary tangles did not appear, and until we can assemble a full chain of events leading from beads to tangles the case, though strong, is still unproven. Secondly, as I’ve discussed previously, research has shown that the plaques can release soluble oligomers into the surrounding neural tissue and will therefore serve as reservoirs of toxic protein even if the fibrils themselves are completely inert.

That Aβ dimers can derange tau regulation in cultured neurons is not a new finding; similar results were reported last year using synthetic dimers (2). Zempel et al.‘s experiments used Aβ concentrations up to 5 μM, but Jin et al. show that naturally-obtained dimers have toxic effects at much, much lower concentrations. As the Zempel et al. paper suggests (consistent with much previous work), dysregulation of calcium levels caused by Aβ oligomers may be how they cause these effects. It is not presently clear why natural oligomers should be four orders of magnitude more potent than the various kinds of synthetic dimers at causing the effect; an understanding of this difference may be crucial in developing a suite of effective treatments for the disease.

1) Jin, M., Shepardson, N., Yang, T., Chen, G., Walsh, D., & Selkoe, D. (2011). Soluble amyloid β-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1017033108 OPEN ACCESS

2) Zempel, H., Thies, E., Mandelkow, E., & Mandelkow, E. (2010). Aβ Oligomers Cause Localized Ca2+ Elevation, Missorting of Endogenous Tau into Dendrites, Tau Phosphorylation, and Destruction of Microtubules and Spines. Journal of Neuroscience, 30 (36), 11938-11950 DOI: 10.1523/JNEUROSCI.2357-10.2010

Oct 112010

ResearchBlogging.orgClostridium difficile is an intestinal pathogen that causes diarrhea in hospitals and other healthcare settings (including nursing homes). Present as a commensal bacterium in a significant fraction of the population, C. difficile is usually rather harmless, its numbers suppressed by competition with the intestinal flora. When its competitors are decimated by antibiotics, however, C. difficile flourishes, releasing toxins that cause inflammation and diarrhea, which can be dangerous because the individuals suffering these effects are often already ill. There has been conflicting information, however, as to which of C. difficile‘s toxins are necessary to cause disease. A paper in the recent Nature (1) aims to resolve the question.

The two best-characterized C. difficile toxins (TcdA and TcdB) have the same general arrangement and function (and ~45% identical AA sequence). An N-terminal glucosylating domain attacks the cytoskeleton of host cells by inactivating Rho GTPases, a C-terminal domain mediates binding and uptake by the host cells, and a protease domain in the middle releases the glucosylating domain to do its work. Since these proteins appear to serve redundant functions, one might expect that both would support virulence. However, preceding work in the field has variously identified TcdA or TcdB as a key virulence factor (2,3). Differences in methodology and materials have contributed to the confusion, in part because different kinds of cells seem to be more or less susceptible to particular toxins, and different strains of C. difficile might have different behaviors.

Kuehne et al. aim to relieve some of the confusion by removing a subset of these confounding factors. In a single strain of C. difficile they inactivated the genes for either TcdA, TcdB, or both by inserting introns into them. An intron would be no problem for a eukaryote, but bacteria can’t handle them, so this has the effect of eliminating the expression of the gene. They then tested the toxin mixtures shed by the bacteria against cultured human and monkey cells. As expected, A-B- bacteria (with both toxins knocked out) showed no toxicity towards the cells, but A-B+ and B+A- variants were toxic towards both kinds of cells to roughly the same degree. This suggests that both toxins are sufficient for virulence.

This implication was largely backed up by a subsequent experiment in hamsters. The animals were dosed with an antibiotic and then infected with C. difficile spores of a single strain. Colonization occurred (in every case but one) within three days. The hamsters infected with A-B- C. difficile remained asymptomatic until the end of the experiment, but the recipients of the other strains all perished within a week. The A+B- group survived somewhat longer, but not dramatically so; again, this supports the interpretation that both proteins are sufficient for virulence.

This contrasts with an earlier study published in Nature (2) where it was shown that deletion of the B toxin protected hamsters from C. difficile-associated disease, using very similar protocols. Kuehne et al. attribute the differences in their results to the hamsters or genetic variation in the C. difficile strains used. While the virulence of the B- strain in this experiment was slightly attenuated, all colonized hamsters still died in relatively short order, and in human beings the situation might well be reversed, since cultured human cells are more vulnerable to toxin A.

The results of Kuehne et al. largely agree with earlier experiments (3) and with what one would naturally expect of two very similar toxins being released by the same organism. While susceptibility to a particular toxin may vary with characteristics of the host species or cell type, it seems likely that both toxins are capable of supporting virulence. While it is to be hoped that additional research will clarify the reasons for the discrepancy between these two experiments, efforts to treat C. difficile-associated disease by attacking the toxins should proceed with the assumption that both must inactivated. Thanks to their functional and sequence similarity this will hopefully not be too much of a complication.

1. Kuehne, S., Cartman, S., Heap, J., Kelly, M., Cockayne, A., & Minton, N. (2010). The role of toxin A and toxin B in Clostridium difficile infection Nature, 467 (7316), 711-713 DOI: 10.1038/nature09397

2. Lyras, D., O’Connor, J., Howarth, P., Sambol, S., Carter, G., Phumoonna, T., Poon, R., Adams, V., Vedantam, G., Johnson, S., Gerding, D., & Rood, J. (2009). Toxin B is essential for virulence of Clostridium difficile Nature, 458 (7242), 1176-1179 PMCID: PMC2679968 OPEN ACCESS

3. Voth, D., & Ballard, J. (2005). Clostridium difficile Toxins: Mechanism of Action and Role in Disease Clinical Microbiology Reviews, 18 (2), 247-263 PMCID: PMC1082799 OPEN ACCESS

Aug 032010
ResearchBlogging.orgMost people never learn about an actual scientific controversy. Almost every “controversy” that bubbles into the public eye is manufactured, often reflecting social or ethical differences rather than genuine disagreements between experts about how different models fit to reality. Actual scientific controversies tend to be highly technical, and often concern points that lay people find to be esoteric. That doesn’t mean that the issues involved aren’t important, or that they’re even difficult to understand. One controversy that has unfolded over the past few years and now may be over relates to a seemingly simple question. Where do adamantane drugs bind to the influenza A M2 channel?

Previously, on As the Channel Twists

Bill DeGrado and James Chou, whose
competing structures began the controversy

The M2 proton channel plays an essential role in the life cycle of the influenza virus. The activity of the channel could be blocked, at least in influenza A, by drugs called adamantanes, including amantadine and rimantadine. Unfortunately, these antiviral drugs have been fading in efficacy due to the spread of an S31N mutation that interferes with their binding. On January 31, 2008, two articles appeared in the scientific journal Nature showing adamantanes bound to the M2 channel. Unfortunately, the structures had different answers about where the drug was bound. The X-ray crystal structure from Bill DeGrado’s group at the University of Pennsylvania placed amantadine in the center of the channel’s pore, suggesting a simple pore-blocking model (PBM) for inhibition. The NMR structure from James Chou’s group at Harvard University located rimantadine on the outside of the channel, ultimately giving rise to an allosteric, dynamic quenching model (DQM) of adamantane activity.

As outlined in my previous post on the M2 channel, there was conflicting functional evidence as to which site was actually relevant in vivo, and reasons to doubt the conclusions from both structures. Since that time, several papers have been published that substantially clarify the issue. At this point, the evidence strongly supports the PBM as an explanation of adamantane activity in vivo.

Sure adamantanes bind there, but does it matter?

The direct observation of NOEs, even weak ones, between the adamantane and the protein proved that the drugs were binding at the DQM site, but there were some significant areas of concern with this finding. The greatest worry was due to the extremely high concentration of ligand used in the NMR experiment. This opened up the possibility that the DQM site was a low-affinity site that would not see binding under normal circumstances. Because both models had explanations for the efficacy of the S31N mutation, the only way to address the question would be to make mutations that would abolish binding at the DQM site and see if adamantanes were still effective. Because aspartate 44 was proposed to form a hydrogen bond to rimantadine, it was thought that a D44A mutation would eliminate binding, and if DQM was true, adamantane activity. This prediction was borne out by an experiment performed in liposomes by the Chou lab (6), but Robert Lamb’s group from Northwestern University was not able to replicate this result in X. laevis oocytes (4).

Robert Lamb has studied the
M2 channel since the 80s.

What Lamb’s group did do was test different parts of the influenza A channel for adamantane sensitivity by fusing them to the adamantane-insensitive influenza B channel. These A/B M2 chimeras should in principle have adamantane susceptibility if the legitimate binding site got imported from A to B. Their first results in this experiment were somewhat inconclusive. Adding the N-terminal portion of the A channel to the C-terminal portion of the B channel (essentially sticking the PBM site into B M2) created a chimera that was somewhat sensitive to amantadine treatment, but the effect was nowhere near what occurred for WT A channel (4). Subsequently, Lamb’s group expanded these experiments to add a little bit more of the N-terminal sequence to the chimera, which then almost perfectly matched the WT A channel’s susceptibility. Notably, when they made the opposite chimera that incorporated the DQM site from A into the B channel, only a very slight inhibitory activity was observed upon addition of amantadine (5). While the conclusions that can be drawn from the chimeras are limited by their particularly odd provenance, the fact that transplanting the PBM site from one channel to another confers adamantane susceptibility suggests that this is the functional binding site.

An upcoming paper in PNAS clarifies the picture somewhat using surface plasmon resonance (SPR) (7). This technique detects the binding of a ligand as a change in physical force exerted by a protein tethered to the surface of a gold chip. In this case, the tethering was mediated by a DMPC liposome. This was a tricky experiment because adamantanes like the greasy portions of lipid bilayers, so they can bind to the liposome itself. Rosenberg and Casarotto, however, were able to control for this effect. Their SPR experiments detect two distinct adamantane binding sites on M2 with vastly different affinities. Rimantadine binding at the high-affinity site could be abrogated by S31N and V27A mutations, but not a D44A mutation. At the low-affinity site, rimantadine binding could be knocked out by a D44A mutation or an S31N mutation, but not a V27A mutation. This result indicates that both binding sites are functional (and, incidentally, that the S31N mutation does indeed exert an allosteric effect on the DQM site). However, the authors note that the adamantane concentrations used in actual treatment are too low to significantly populate the low-affinity site, given the dissociation constants they calculated. This argues that the DQM site is irrelevant in vivo.

Amantadine caught in the pore

Mei Hong has studied M2
extensively by NMR

One of the problems with the PBM was that the crystal structure that supported it was unsatisfactory in a variety of ways. The structure was made using a construct that consisted of only the transmembrane segment of the protein. This construct could not be reconstituted in micelles, and functional experiments showed that it was not very similar to the WT in terms of its activity. In addition, the extra electron density in the pore could not be unambiguously assigned as amantadine. In a paper from February of this year, the DeGrado group collaborated with Mei Hong’s group at Iowa State University to produce a structure of amantadine bound to M2 using solid-state NMR (2). An important advantage of this approach is that one can take spectra of proteins embedded in a membrane without penalty, because there is no requirement for the protein to tumble freely. While there are some trade-offs in terms of resolution and the kinds of data that can be obtained, biomolecular solid-state NMR can help us answer some very tricky questions.

Amantadine (yellow) in the
pore binding site.

The approach proved to be especially fruitful here. Cady et al. used a technique that allowed them to determine whether the amantadine was near a particular residue, by labeling residues with 13C and the amantadine with 2H. If a 13C nucleus is coupled to a 2H nucleus by a dipolar interaction, a pulse that dephases the 2H nucleus will affect the 13C nucleus in a distance-dependent manner. When Cady et al. made samples at a ratio of one amantadine per channel, they found that the signal from S31 was significantly broadened, but that from D44 was not, proving that the amantadine is close to S31 under these conditions. The D44 signal was affected at higher amantadine concentrations, but never to the same degree as the S31 signal. Other residues at the PBM site were also affected by the presence of amantadine. Using an alternative version of the REDOR experiment, Cady et al. were able to generate distance constraints and, in combination with other data, generate a structure for the channel with amantadine bound (see their figure at right) (2). Note the residues that are close to the amantadine in this structure: V27, S31, G34, and H37. This will be important in a minute.

The Cady et al. paper has several advantages over the DeGrado group’s original crystal structure. The first, and most important, is that the protein was reconstituted in DMPC vesicles rather than OGP bilayers. The lipids themselves, and the vesicle structure, more closely mimic the likely environment in vivo than the crystal conditions, and they are also more biologically-relevant than the DHPC micelles employed in Chou’s original determination. In addition, the pH of 7.5 matches the experimental conditions used by Chou and allows for a direct comparison of results under conditions where the channel’s conformation should in principle be the same. The REDOR results provide unambiguous evidence that amantadine binds preferentially in the pocket for this construct. Their observation that amantadine has approximately 100-fold higher affinity for the PBM site, but can also bind the DQM site, agrees nicely with the functional data, especially the SPR results.

Support for an allosteric mechanism?

Bob Griffin,
SSNMR master

However, Cady et al. still used the truncated construct that possesses significantly altered activity relative to WT, which brings us to an upcoming paper in JACS by Andreas et al. (1). Chou’s group collaborated with Robert Griffin’s group at MIT to map the chemical shifts of a somewhat larger construct of M2 that is known to have relatively normal activity. The longer construct is also known to form a fairly stable tetramer, and this may have improved its spectral properties. The construct was inserted into membrane bilayers that were lyophilized for the NMR experiment, with and without rimantidine present. The chemical shifts of 15N and 13C nuclei of residues in the transmembrane helices were compared between these two states as a way of localizing the adamantane binding site. As Andreas et al. show in their figure 2 (recapitulated below in a slightly altered fashion), the chemical shift changes in response to the binding event are quite widespread. The authors argue that this observation favors an allosteric inhibition mechanism. That may well be true, in a sense, but unfortunately that does not mean that the observation favors DQM.

The binding of a ligand generally alters the chemical shifts of a protein by changing its structure, reshaping the arrangement of bond angles that dictates the electron distribution around the relevant nuclei. It is of course possible that a small change in the protein’s conformation at the binding site propagates into a large change elsewhere, but this is not typically observed. The most reasonable interpretation of chemical shift changes (Δδ) upon ligand binding is that the largest shifts observed are nearest to the ligand, while the smaller shifts are farther away. The original version of figure 2 presents the data in a fairly simple way, and does not distinguish very large changes from relatively small ones. So, I made a new version of the figure for you, where I’ve taken the additional step of scaling the color according to the magnitude of Δδ.

M2 channel showing Δδ
due to rimantidine binding

This adaptation of the Andreas et al. figure, shows the NMR micelle structure (PDB code: 2RLF) with purple rimantadine at the DQM binding site and the helices colored in blue according to the Δδ. This was done very roughly, by eyeballing the graphs to the left in figure 2, scaling the Δδ based on the nucleus measured, and adding it all up across all three nuclei. The more intense the blue, the larger the Δδ. It should be immediately apparent that the largest chemical shift changes are located a substantial distance from the DQM site (although there are some smaller changes near that site). What’s perhaps less immediately obvious is that the largest chemical shift changes belong to residues located in the pore. To make this point more clear, check out the figure below and to the left. This is the same structure, that I have now tilted so we are looking down the barrel of the channel. The side chains of the four residues with the largest chemical shift changes (V27, S31, G34, H37) are shown in light green for contrast (obviously there’s nothing for G34). Three of them are sticking into the pore in this model (G34′s Cα faces the pore); the fourth is S31. You might recall that the structure from Cady et al. (above) puts all four of these large Δδ residues right next to the ligand.

The largest chemical shift changes in response to rimantidine binding occur near the proposed PBM site and far from the DQM site, and the residues most affected are those facing the pore. Andreas et al. argue that this information is insufficient to positively localize the drug, due to the large chemical shift change at H37 Cα, but this doesn’t seem particularly convincing. The concentration of large chemical shift changes at the N-terminal end of the channel strongly argues for the ligand binding in this region. As for the significant Δδ at H37, the observations could quite possibly be due to ring-current effects from the repositioning of the adjacent tryptophan side chains (light red in the figure to the left). We know that binding of adamantanes has at least some effect on W41 thanks to Czabotar et al. (3), who measured adamantane binding by observing changes in intrinsic tryptophan fluorescence. Of course, changes in fluorescence are a very general indicator of structural or dynamic change, but the previous finding supports the possibility of interpreting the H37 observations as side-chain effects rather than ligand proximity.

In contrast, there is no such explanation for the significant chemical shift changes at the N-terminal end of the channel, which has no aromatic residues. The significant Δδ in this region must be due to rearrangements of these residues themselves, rather than long-range effects or ring currents. Thus, the most plausible model explaining these data remains one in which adamantanes bind near V27 and S31, propagating some kind of structural change to W41, rather than the other way around. In this regard, I agree that the data from figure 2 establish that adamantane binding has an allosteric effect. These data, however, do not support the DQM Chou proposed previously.

Conclusions, Lessons

The DQM hit its high-water mark with the Pielak et al. PNAS paper back in 2009 (6), in which the model’s prediction that the D44A mutation would significantly alter adamantane sensitivity was borne out by experiments in liposomes. Since that time, however, the evidence has started to weigh mostly against it. The D44A results could not be replicated in X. laevis oocytes, and lovely chimera experiments in this system demonstrated that the N-terminal region of M2 was critical for adamantane sensitivity (4)(5). Live viruses remained sensitive to adamantanes even if they were reverse-engineered to have the D44A mutation. Rosenberg and Casarotto showed that the D44A mutation only affected binding at absurdly high rimantadine concentrations (7). Finally, the Cady et al. study provided unambiguous evidence for adamantane in the pore of the channel (2). In light of these findings, only a crystal-clear result in favor of the DQM could really save it.

Although their findings convincingly illustrate an allosteric effect from rimantidine binding, Andreas et al. do not provide that result. Even their own chemical shift data seem to support the PBM model. Of course direct dipolar couplings would provide a totally unambiguous answer as to the location of the rimantidine, but in light of our existing knowledge about the system, that experiment doesn’t seem necessary. At this point there is no serious reason to doubt that the physiological inhibition of M2 channel results from adamantane drugs binding to the pore.

The papers cited in this article represent decades of man-hours and significant amounts of money spent in resolving what might seem like an esoteric point. Given the enormous effort that went into resolving the  seemingly simple question, you might be tempted to ask what went wrong. The answer is, “nothing”. This is how the scientific process is supposed to work. Two groups came at the same problem in different ways and got different answers, which is hardly a surprise because no experiment is perfect. More experiments were carried out to determine which model best represented the physical reality. Eventually, the weight of the evidence strongly supported one model over the other. The best data we have right now really point to a single conclusion. The process succeeded, and nobody needed a superior court judge or a congressional hearing.

That doesn’t mean we can’t take some lessons from the experience. Most prominent among these is that we must have serious reservations about NMR structures derived from proteins bound to or inserted in micelles. What we know about the M2 channel tells us that adamantanes prefer to bind in the pore. That they did not do so (or at least, could not be detected doing so) in the micelle-based structure suggests that something about the micelle itself made that impossible. We know that the forces exerted on proteins by membrane curvature can be substantial, and the structure of a micelle is very unlike the structure of a cell membrane. Solution NMR in bicelles may yet prove to be a superior approach for some systems, but in this case it was solid-state NMR that provided the vital evidence. Solid-state has its own set of limitations, but it’s clear proper membrane context is absolutely vital to getting good answers about membrane protein structure and function.

Knowing the actual binding site of adamantanes may prove to be very important in aiding the design of alternative drugs that achieve the same inhibition of the channel. The papers from the DeGrado and Hong groups have already made several interesting recommendations in this regard. Even what has been learned about the remote site may not be fruitless. Though it is not the source of the physiological activity of adamantanes, several experiments have made it clear that there is some kind of allosteric interaction between S31 and the DQM site. It may be possible to attack M2 through this site with a specifically-designed high-affinity drug, even if adamantanes themselves don’t work this way.  If that proves to be the case, then this will be the best kind of scientific controversy: one where we learn something important from both sides.


(1) Andreas, L., Eddy, M., Pielak, R., Chou, J., & Griffin, R. (2010). “Magic Angle Spinning NMR Investigation of Influenza A M2: Support for an Allosteric Mechanism of Inhibition.” Journal of the American Chemical Society DOI: 10.1021/ja101537p

(2) Cady, S., Schmidt-Rohr, K., Wang, J., Soto, C., DeGrado, W., & Hong, M. (2010). “Structure of the amantadine binding site of influenza M2 proton channels in lipid bilayers.” Nature, 463 (7281), 689-692 DOI: 10.1038/nature08722

(3) 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

(4) 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, 105 (31), 10967-10972 DOI: 10.1073/pnas.0804958105 OPEN ACCESS

(5) Ohigashi, Y., Ma, C., Jing, X., Balannick, V., Pinto, L., & Lamb, R. (2009). “An amantadine-sensitive chimeric BM2 ion channel of influenza B virus has implications for the mechanism of drug inhibition.” Proceedings of the National Academy of Sciences, 106 (44), 18775-18779 DOI: 10.1073/pnas.0910584106

(6) 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, 106 (18), 7379-7384 DOI: 10.1073/pnas.0902548106

(7) Rosenberg, M., & Casarotto, M. (2010). “Coexistence of two adamantane binding sites in the influenza A M2 ion channel.” Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1002051107