|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?
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.
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