Zombie cyclophilins catalyze HIV capsid isomerization

If you're going to study the role an enzyme plays in a biological pathway, it's often useful to "kill" it with a mutation. For example, the proline cis-trans isomerase cyclophilin A (CypA) needs a particular arginine residue for its chemistry, so mutations that remove or alter that functional group, like R55K and R55A, should destroy the protein's function and have effects on the related pathways that help illustrate its role. The hydrophobic pocket it uses to bind substrates is made by residues like H126, F113, and W121. Growing or shrinking those residues should alter the shape of the pocket and change binding or activity, leaving the enzyme "dead".

Using model reactions and various binding assays, researchers have previously examined a number of these mutants (4,7) and found that they diminish isomerase activity and alter inhibition. However, a detailed study of the effects of the mutations on CypA's catalytic cycle has not been performed. Former Kern lab members Daryl Bosco (now a professor at UMass Medical) and Elan Eisenmesser (now at UCHSC) examined these mutants in greater detail to see how they really behaved. I also contributed some data at the last minute, when the third reviewer requested we study an additional mutant, prompting a scene that I promise was not too much like that Downfall parody. In every case we found that these enzymes, although significantly impaired, weren't as dead as they had seemed.

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Prion diseases: protein is enough

Spongiform encephalopathies are transmissible diseases that can have a major economic impact on agricultural exports, and pose a significant challenge for surveillance of the food supply. Scientists generally believe that these diseases are transmitted via a self-propagating, aberrant conformation of the prion protein (PrP). This prion hypothesis suggests that PrP alone should be sufficient to cause symptoms or death. If this hypothesis is true, then it should be possible to reproduce the disease using recombinant proteins expressed in yeast or bacteria. In tomorrow's Science, researchers from Columbus and Shanghai report that they have managed to do this, establishing that PrP alone can account for prion disease transmission.

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Don't look for "the" structure

It surprises me how often I hear students, postdocs, and even professors talk about determining the structure of a protein. A singular structure has the advantage of being relatively easy to interpret, but the cost of this is often the loss of functional data. It's easy to understand how this terminology emerges from the discipline of crystallography, which after all only works when the protein molecules adopt only a small number of conformations. Yet even when it comes to NMR, a technique that should be very sensitive to the fact of structural multiplicity, the language of researchers and the structural tools available to them are too often oriented towards the idea of a singular structure. But any representation of a protein as a single conformation is a simplification — every protein exists in multiple structural states.

Trivially, we are aware that a given polypeptide chain can adopt a number of different conformations — the "folded state" of any given polypeptide chain covers only a tiny sliver of the possible conformational space. A protein that is "unfolded" occupies not a single, well-defined state but a vast multiplicity of states, and this kind of statement is not controversial because we tend to imagine unfoldedness as a messy chaotic jumble of conformations. The reality is less cut-and-dried: although unfolded proteins may have no regular structure, many still have a propensity to form particular secondary structures or interactions. The reality of denatured proteins is that they have a complex and varied energy landscape, not an array of possible structures that all have roughly equivalent energy. The flipside of the popular view is that the a protein's native state draws down to a sharp energy well, and this conception is also misguided.

The most dramatic counterexamples to the idea of a neat, punctate energy well come from proteins that adopt several different folds in the native state. One relevant case is lymphotactin, which freely interconverts between an α/β monomer and an all-β dimer under physiological conditions. Lymphotactin may be unusual, but the principal message from that study is one that ought be paid attention to in others, particularly when the protein in question has functional conformational diversity. Consider α-synuclein, a protein implicated in Parkinson's disease. In the presence of some detergent micelles this protein is known to take on an α-helical hairpin structure, with two helices laying down on the charged surface of the lipid headgroup. In solution, however, it seems to take on a number of different forms, and may interact with true lipid bilayers in a completely different way than it interacts with micelles. For proteins that interconvert between several different physiologically-relevant folds, one is never pursuing the structure, but rather a structure.

Of course, we don't expect most proteins or domains to regularly adopt alternate overall folds. However, reorientations of domains or monomers is a relatively common behavior, and one that poses a sticky challenge for structural biologists because incidental properties of a particular arrangement may bias our experiments towards observing it. A minor member of the ensemble, if it has favorable packing geometry, may exclusively populate a crystal. Similarly, NMR experiments to determine domain arrangement via residual dipolar couplings must always be undertaken with an eye to ensuring that interactions with the aligning media do not bias the results. No single structure of adenylate kinase can instruct us about its catalytic cycle, and structures of the unbound state do not capture the reality that the protein continues to open and close in the absence of ligand. Single structures do not capture motions of domains or monomers relative to each other and that often means an incomplete understanding of function.

Domain motions are also an overly dramatic example, because simpler rearrangements of the backbone take place in many proteins, even when regular secondary structures are evident. Fluctuations of the main chain play a functional role in several proteins — as, for instance, in the flaps of the HIV protease. Additionally, rearrangements of the backbone have a significant role in signaling, as in NtrC, which I'll talk about more in two weeks. Proteins where the main chain rearranges in response to ligand binding or post-translational modification generally cannot be described by a single structure.

Even if the backbone is rigid, every protein will have flexibility in the side chains of its amino acids. One of course expects to see this kind of behavior in side chains on the surface of a protein, where it is usually dismissed as irrelevant. However, we also know that side chains can rotate and move in the core of a protein, and that on some protein surfaces they can undergo coherent rearrangements. I'll talk a bit more about the functional relevance of side-chain motions next Thursday. For now, suffice to say that side chain rotations cannot be so easily ignored and sometimes have functional effects. Structural studies that do not capture these rotations may be missing something important.

My point here is not that single structures are stupid or useless. A structure can be very informative about about a protein's function, and often has great power to explain the effects of mutations and ligands. However, we should not mislead ourselves into thinking that any single structure will have all the answers, or indeed any of them. Every protein is a constantly interconverting ensemble of structures, and there are many layers of structural diversity within that ensemble, reaching from whole fold rearrangements to "mere" side-chain adjustments. Determining the structure of a protein is not a coherent goal for a research program. The successful structural biology study will characterize the conformation and energy of key, functionally-relevant members of the protein's structural ensemble and identify the pathways between them.

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Proteins stick together when it's crowded

One of the many ways that a living cell does not resemble a test tube is in the degree to which its internal environment is crowded. Cells are crammed full of massive protein complexes, vesicles, organelles, carbohydrates, peptidoglycan, and other assemblies that occupy a great deal of space. The tubes and cuvettes used for biochemical experiments, in contrast, typically contain nothing more than a few proteins and small molecules of interest along with a relatively dilute set of salts and buffering agents. Because many complexes appear to be stabilized by crowding, and excluded volume effects are known to favor compact, folded protein chains, there is considerable interest in estimating how protein behavior changes in the spatially-restricted environment of the cell. In some cases this is approached using NMR to study changes in dynamics and structure inside cells. In a recent issue of Biophysical Journal, groups from Florida State University and Rehovot University, in separate papers, address how crowding affects protein-protein interactions.

Both groups perform biochemical experiments in vitro to examine this question. In order to crowd the solution, they add reagents such as ficoll, polyethylene glycol (PEG), and dextran, and compare the changes in binding and kinetics to solutions that have merely been made more viscous (through the addition of glucose, for instance). Batra et al. study the association of two components of the E. coli DNA polymerase III and find that the presence of crowding agents slightly stabilizes the complex. However, as the size of the crowders is increased, this stabilization is diminished. Batra et al. develop a relatively simple mathematical model that suggests this observation results from the fact that larger particles pack less efficiently, leaving larger "holes" in which the protein complex sees something more like dilute solution.

Phillip et al. study several protein complexes. Similar to Batra's group, they find that crowding with dextran modestly increases the binding affinity of two of their protein pairs, but that this is not replicated for crowding with PEG. Particularly for PEG-1000, there was a clear decrease in affinity, although a non-crowding viscogen (ethylene glycol) had an even greater effect. Phillip et al. also measured the kinetics of binding, and found that the association rates were significantly lower in crowded solutions, as compared to buffer. However, when the rates were corrected for the effect of viscosity, it appeared that the crowding agents slightly increased the association rate. The authors attribute this to excluded volume effects in the binding transition state. The dissociation rate was also slightly reduced in crowded solutions, which the authors explain by the longer lifetime of the encounter complex (allowing a larger fraction of complexes to fall back to the lower-energy bound state).

Given that crowding appears to have a profound effect on the function of certain complexes, the relatively small effects observed in these studies might seem confusing. Batra et al. argue that although each individual binding interaction is only modestly stabilized, the effect should be cumulative. As a result, multi-subunit complexes will experience a greater effect than small heterodimers. Additionally, the most famous examples of crowding enhancement involve very large complexes — the ribosome, decameric assemblies, hemoglobin polymers, etc. In comparison, the complexes formed in these model studies are quite small. It may be that the stabilizing effect of crowding depends to some degree on the size of the complex to be formed. While similar size is difficult to achieve in strictly heterodimeric systems, it should be possible to monitor the assembly of large complexes like GroES/GroEL under crowded conditions. A study of the relationship between the size or number of components in a complex and crowding stabilization may prove instructive.

Batra, J., Xu, K., Qin, S., & Zhou, H. (2009). Effect of Macromolecular Crowding on Protein Binding Stability: Modest Stabilization and Significant Biological Consequences Biophysical Journal, 97 (3), 906-911 DOI: 10.1016/j.bpj.2009.05.032

Phillip, Y., Sherman, E., Haran, G., & Schreiber, G. (2009). Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions Biophysical Journal, 97 (3), 875-885 DOI: 10.1016/j.bpj.2009.05.026

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How we taste umami

Although we still do not know the full breadth of our flavor-sensing capabilities, human beings are known to possess receptors for at least five basic tastes. Probably you have known about the sweet, sour, salty, and bitter flavors since you were in grade school, but the fifth, umami, was less widely accepted in the West until recently. Umami is a savory flavor element that is found in many foods, including tomatoes, parmesan cheese, truffles, and many kinds of meat and seafood. The umami taste primarily detects the amino acid glutamate (hence the popularity of the food additive monosodium glutamate, or MSG), but the effect is also intensified by the presence of the nucleotide inosine monophosphate (IMP). In a recent (open access) paper in PNAS, researchers from two corporations examined the umami taste receptor to understand how this happens.

The umami flavor is detected by a pair of G-protein coupled receptors (GPCRs) that have an external venus flytrap (VFT) domain in addition to their classic 7-helix trans-membrane domain (TMD). This complex is closely related to the sensor for the sweet flavor: in fact one of the receptors (called T1R3) is the same in both sensors. It is the second receptor (T1R1 for umami, T1R2 for sweet) that determines what taste is recognized. What we don't know for sure is whether it is the TMD or the VFT of these receptors that identifies the flavor component.

In order to answer this question, the researchers performed an experiment known as a "domain swap". Using recombinant DNA technology they assembled two chimeric proteins, one with the VFT of umami and the TMD of sweet, and one with the VFT of sweet and the TMD of umami. They then inserted these proteins into cultured cells that would fluoresce when the receptors were activated. The authors suspected that the VFT is primarily responsible for binding the ligand. As you can see from figures 1 & 2 (this is an open access paper, so go ahead and take a look), the experiment bears this out. The chimera with the VFT of sweet caused a fluorescent response in the presence of compounds such as sucrose and aspartame, while the umami-VFT chimera reacted to glutamate and aspartate. You can also see in figure 2C that the presence of IMP dramatically enhanced the activity of glutamate in this chimera. This indicates that the VFT is also responsible for IMP synergy in the umami receptor.

The hurdle in going further than this is that no structure of the umami VFT is available, which makes it difficult to figure out exactly how everything fits together. However, T1R1 has a close evolutionary relationship to the metabotropic glutamate receptors (mGluR), and a crystal structure of that VFT is available. Using conserved and homologous residues as a guide, the authors made a model of the T1R1 fold from the mGluR data. Based on this model they predicted certain amino acids that would be essential for glutamate binding in T1R1 and then mutated them in order to measure the effect. Residues that were predicted by the model to interact with the zwitterionic amino acid backbone proved to be essential for ligand recognition. Interestingly, the amino acids that contact the side-chain carboxylic acid of glutamate in mGluR are not conserved in T1R1, and mutations at the matching sites do not alter glutamate binding. However, these mutations eliminate the effect of IMP.

In order to understand this behavior, the authors modeled the binding cleft in the closed state, with IMP and glutamate in place. Glutamate binds at the bottom of the cleft, with its side chain pointed outwards. This conformation puts several positively-charged residues from the two lobes of the VFT close together higher up in the cleft. The authors propose, in keeping with previous models of VFT behavior, that the binding of the glutamate lowers the energy barrier between the open and closed states of the domain, but that glutamate alone is not sufficient to hold the domain closed. Their model places IMP higher up in the cleft, where its negatively-charged phosphate interacts with the positive residues. Thus, IMP stabilizes the closed conformation of the VFT domain.

Some more work here would be welcome, particularly in the form of experimental crystal structures of the T1R1 VFT that can confirm the homology model. The VFT is rather large, but using a perdeuterated sample in a high-field magnet it might be possible to confirm the population-shift mechanism using NMR experiments. Lower-resolution techniques such as FRET may also be able to catch this stabilization behavior. If the model proves to be accurate, it would serve as an interesting example of positive allostery from a population shift.

Although these experiments only concerned the umami taste receptor, this allosteric mechanism may be a more general feature of certain GPCRs. The authors indicate that they have unpublished data showing similar behavior in the sweet receptor, and it may be possible to design an allosteric stabilizer for any GPCR with a VFT domain. Because the related mGluR receptors are involved in many neurological and psychological diseases, successful design of such activators may have some therapeutic value.

F. Zhang, B. Klebansky, R. M. Fine, H. Xu, A. Pronin, H. Liu, C. Tachdjian, X. Li (2008). Molecular mechanism for the umami taste synergism Proceedings of the National Academy of Sciences, 105 (52), 20930-20934 DOI: 10.1073/pnas.0810174106 OPEN ACCESS

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Where do new enzymes come from?

Biochemists 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

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An enzyme with a monkey's tail

It 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.

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NSAIDs vs. Alzheimer's: Multiple modes of action?

Loads of interesting stuff is going on in Alzheimer's research right now. While the hot news is about a trial showing significant benefits from going after tau tangles, a recent paper in PLoS ONE continues to investigate the pathology of the amyloid-β peptide. As I've mentioned in previous posts, cleavage of the amyloid precursor protein to a 42-residue peptide (called Aβ1-42 in this paper) initiates the formation of peptide oligomers and eventually plaques. Recent research has indicated that these oligomers are sufficient to cause the development of Alzheimer's disease, but the mechanism by which they do so remains uncertain. Sara Sanz-Blasco and colleagues show that Aβ oligomers disrupt calcium homeostasis in neurons, damaging the mitochondria and promoting apoptosis, and that certain NSAIDs can suppress these adverse mitochondrial effects (1). PLoS ONE is open access, so go ahead, open the article up in another window, and follow along.

Although the appearance of plaques and neuronal death are classic hallmarks of Alzheimer's pathology, the relationship between these features is not well understood. For instance, it is possible the plaques themselves kill neurons or impair neural function. However, it seems equally likely that the appearance of plaques and the death of neurons are two distinct effects with a single cause. This view is supported by the oligomer toxicity study, but that study fails to resolve the question of exactly how Aβ oligomers kill neurons. Previous work has associated Aβ with derangement of cellular calcium (Ca²⁺) management — a 2005 paper by Demuro et al. (2) showed that soluble Aβ induced an increase in intracellular Ca²⁺ in a neuroblastoma cell line. Sanz-Blasco et al. therefore decided to directly test whether Aβ oligomers were increasing Ca²⁺ levels in neurons, and specifically in mitochondria. In order to do this last bit they used a low-affinity aequorin targeted specifically to mitochondria.

Allow me digress... to many of my readers that probably sounds like a terrible idea. If you're trying to detect a particular chemical in the cell, it seems like the best thing to do would be to get a high-affinity binding partner. And if figuring out whether there is any calcium in the mitochondria is what you want to do, then a high-affinity detector makes sense. However, when you're using a small amount of a sensor to detect changes in the concentration of a large amount of ligand, a low-affinity sensor is what you want.

To see why, take a look at the graph on the right. This is just a rough calculation based on a situation where the detector is at a concentration of 100 µM and the concentration of its ligand (that you're trying to detect) changes from 10 mM to 100 mM. Note that the concentration of the detector is at most 1% that of the ligand. If the dissociation constant K_D of this complex is 1 mM (blue) (a lower K_D means higher affinity), then the detector is almost saturated when you start, and the percentage occupied doesn't change very much over the course of the experiment. This means that it will be very difficult to tell the difference between, say, 50 mM ligand and 100 mM ligand, because that amounts to a signal difference of 1% of the maximum response. The situation gets a little better if the K_D is 10 mM (green). The lowest affinity detector here (K_D = 50 mM, red) actually does the best job of distinguishing between 50 mM and 100 mM ligand, because the difference in response amounts to 17% of the total dynamic range. Ideally, you want to tune the K_D of your detector in such a way that its response to changes in ligand concentration is large and linear over the range you are likely to be observing. For the last detector, this range lies between 10 and 40 mM of ligand, so that would likely be the ideal range to investigate with it.

The precise numbers are different in the present paper, but the principle is the same. The affinity you want in your detector will depend on what you are trying to detect and the circumstances under which you are trying to detect it. In this case, the researchers are trying to measure changes in calcium ions over a fairly wide range, which have a pretty high concentration in mitochondria, and they're doing it using a luminescent protein, which isn't very concentrated. As a result, a relatively low-affinity detection system is best.

So, what did they find? The results in Figure 1 show that Aβ oligomers and fragments cause an influx of calcium into the cytoplasm of cultured neurons, but preparations of Aβ fibrils did not cause this effect. Moreover, exposure of the cells to Aβ oligomers caused a clear influx of calcium into the mitochondria (Figure 3). This is a problem for a cell because Ca²⁺ overload in mitochondria can cause programmed cell death, or apoptosis. Using the classic TUNEL assay, the authors of this study showed that the Aβ oligomers caused apoptosis. In addition, they showed that treatment with the oligomers caused the release of mitochondrial cytochrome c (a step in the apoptotic pathway) and that the addition of cyclosporin A, which inhibits the release of proteins from the mitochondrion, blocked cell death (Figure 4). Together, these pieces of evidence support the idea that Aβ-induced Ca²⁺ influx into the mitochondria activates the apoptotic cascade, leading to neuronal death. These results are consistent with a very cool study published this week in Neuron (3) showing that amyloid plaques correlated with high neuronal Ca²⁺ levels in vivo (in live mice).

On its own this is pretty interesting, but Sanz-Blasco et al. push it a bit further. Because they had shown previously that some NSAIDs prevent mitochondrial Ca²⁺ uptake in a cancer cell line, they decided to find out if they would work in this instance, too. As you can see from Figure 6, the three NSAIDs tested kept the mitochondria calcium-free, even if the cells were treated with Aβ oligomers. NSAIDs also prevented cytochrome c release and cell death (Figure 8).

Some readers may recall that Kukar et al. showed that certain NSAIDs prevent oligomerization of Aβ1-42, hinting at a possible explanation of these results. However, the controls performed by Sanz-Blasco et al. show that under the conditions of these experiments the NSAIDs they used have no effect on cytosolic Ca²⁺ concentrations (Figure 7). If it is amyloid oligomers that let Ca²⁺ through plasma membranes, then this would appear to rule out structural disruption as a mechanism. Instead, Sanz-Blasco et al. propose that these NSAIDs specifically alter the polarity of the mitochondrial membrane in such a way as to prevent Ca²⁺ uptake.

If this is true, then NSAIDs may be able to perform a double-whammy on Alzheimer's disease. On the one hand, they appear to be capable of altering Aβ cleavage patterns to reduce the formation of toxic oligomeric precursors. In addition, they appear to have an ability to block mitochondrial breakdown and subsequent apoptosis directly. While this is encouraging, and speaks to the value of pursuing refinements of existing NSAIDs as possible Alzheimer's treatments, this experiment doesn't necessarily prove any therapeutic value. Even if the neurons are saved from death, the calcium flood may impair their function to such a degree that their continued survival doesn't matter. Only clinical trials and further research can firmly establish whether current or optimized NSAIDs can provide significant protection against Alzheimer's disease.

1. Sara Sanz-Blasco, Ruth A. Valero, Ignacio Rodríguez-Crespo, Carlos Villalobos, Lucía Núñez (2008). Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs PLoS ONE, 3 (7), 0-0 DOI: 10.1371/journal.pone.0002718 OPEN ACCESS

2. A. Demuro, E. Mina, R. Kayed, S.C. Milton, I. Parker, C.G. Glabe (2005). Calcium Dysregulation and Membrane Disruption as a Ubiquitous Neurotoxic Mechanism of Soluble Amyloid Oligomers Journal of Biological Chemistry, 280 (17), 17294-17300 DOI: 10.1074/jbc.M500997200 OPEN ACCESS

3. K Kuchibotla, S Goldman, C Lattarulo, H Wu, B Hyman, B Backsai (2008). Aβ Plaques Lead to Aberrant Regulation of Calcium Homeostasis In Vivo Resulting in Structural and Functional Disruption of Neuronal Networks Neuron, 59 (2), 214-225 DOI: 10.1016/j.neuron.2008.06.008

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Why are Xfaso and Pfl Cro so different?

A few weeks ago, when I posted on the transitive homology studies performed by the Cordes group, I promised a closer look at the structures when they became available. If you'll recall, one of the central findings of the Roessler et al. paper (1) was that the Xfaso and Pfl 6 Cro proteins, though they had 40% sequence identity, as well as an identical function, had very different structures and dimerization characteristics. The Pfl6 Cro structure is now available in the PDB, and Dr. Cordes was kind enough to send me the Xfaso Cro structure, which has been held up by some technicalities. I made the overlay of the structures to the left, with Xfaso in dark green and Pfl 6 in crimson. As you can see, the N-terminal helix-turn-helix motifs of the two molecules overlay very precisely, with some slight differences in orientation in the context helices. The C-terminal portions, of course, are completely different. How did they get to be this way?

Well, I have a few thoughts. To achieve a significant change in structure like we have here, two possibilities suggest themselves. We can destablilize one structure, or we can stabilize the other. So let's try to look at this from both angles. First, the Xfaso structure, which is on the left. The ribbon is orange for residues that don't change between the two proteins, green for mutated sites, and red over a deleted range. I've also drawn in a couple of the mutated side chains that might have an effect. For instance, at the upper right you can see a glutamate of Xfaso Cro that becomes a glycine in Pfl cro. The presence of the glycine may destabilize the helix. Lower on the helix, a solvent-exposed arginine becomes a hydrophobic leucine in Pfl; likely the structure will change to reduce the contact of the leucine with water. By the same token, a partially-buried threonine at the base of the helix gets mutated to glutamate. Not only might this put an unsolvated negative charge inside a hydrophobic region, but favorable helix-capping interactions of the threonine might be broken. Roessler et al. also point out that a pair of cysteines in the Xfaso structure are in a favorable position to form a disulfide bond; both are absent from the Pfl6 sequence.

On the other side of things, what mutations are stabilizing the new fold of Pfl 6 Cro? Check out the image below, color-coded the same way as the previous structure:
There are a couple of things going on here. First, the hydrophobic residues. The leucine that was an arginine in Xfaso Cro has indeed been buried, up at the top of the structure. Nearby, the glutamate that was a partially-buried threonine in Xfaso is now fully solvent-exposed. In the same vein, an aspartate has become an isoleucine near the middle. This new isoleucine appears to pack into the core of the companion protein near a conserved isoleucine (and a new methionine), and probably accounts to some degree for the stability of the dimer. There are a wealth of minor effects too—an alanine to arginine mutation has created a charged group that is solvent stabilized on one of the strands; down near the bottom a proline-to-arginine change probably has allowed the extension of a helix. Some other side-chains are drawn that I was taking a look at, but probably aren't critical, and of course I've probably missed a few that matter.

The R→L, T→E, and D→I mutations probably do a great deal to change the most stable conformation from all-α to α/β and strengthen dimerization in solution. However, just from looking you can never be sure what mutations are the most important. Doubtless the Cordes lab is already examining which residues are critical in altering the conformation. It will be interesting to see whether point mutations tend to move Xfaso from the all-α to the mixed structure, or whether they produce molten globules.

1. Roessler, C.G., Hall, B.M., Anderson, W.J., Ingram, W.M., Roberts, S.A., Montfort, W.R., Cordes, M.H. (2008). Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds. Proceedings of the National Academy of Sciences, 105(7), 2343-2348. DOI: 10.1073/pnas.0711589105

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Dynamics and tunneling in soybean lipoxygenase

Because even the simplest case of enzymatic catalysis involves multiple steps (i.e. association, chemistry, and dissociation), semantic issues have obscured the question of what role protein structural dynamics play. For instance, the opening of the lids in Adenylate kinase appears to be rate-limiting, but there is as yet no evidence that dynamics play any role in the phosphotranfer. Thus, one can argue that dynamics are critical (because they are rate-limiting) or irrelevant (because they contribute nothing to the chemical step). Some authors, Arieh Warshel for example, have argued that the latter finding is general—that the highly successful transition-state stabilization model leaves no place for dynamics in chemical catalysis. Some researchers, however, believe that dynamics may play a significant role in chemistry when catalysis proceeds by other routes, especially hydrogen tunneling. In this vein, Matthew Meyer, Diana Tomchick, and Judith Klinman presented evidence in last week's Proceedings of the National Academy of Sciences that protein dynamics play a significant role in the chemical step of catalysis for the protein soybean lipoxygenase (SLO-1).

Hydrogen tunneling refers to the idea that a hydrogen may in some instances "tunnel through" an energy barrier, going directly from substrate to product without wasting any time or kT in actually surmounting that barrier. A reaction that primarily reflects tunneling should have three properties. The reaction rate should vary only weakly with temperature, because tunneling mostly divorces the rate from kT. Using an Arrhenius plot, this translates to a low calculated energy of activation. Replacing the reactive hydrogen with deuterium should enormously inhibit the reaction—a large kinetic isotope effect (KIE)—because the doubling of mass makes tunneling less likely. Nonetheless, the difference in the energies of activation (ΔE_a) for hydrogen and deuterium calculated from an Arrhenius plot should also be small, if tunneling is the main mechanism. Conceivably an enzyme could enhance the rate of such a reaction by using dynamics to promote favorable vibrational modes for the tunneling to occur, or to temporarily adopt a disfavored structure that puts reactive groups at a better distance for tunneling.

SLO-1 exhibits all three features, and so Klinman's group has used it as model system to try and understand whether and how enzymes promote tunneling reactions. In this case they have performed a series of mutations of isoleucine 553. In addition to existing data on WT and I553A, they analyzed the KIEs, crystal structures, and activation energies of I553L, I553V, and I553G mutants. They find that the protein as a whole is not much distorted by any of these mutations, nor do any of them appear to have significant effects on the binding pocket or substrate dissociation constant—the K_d for each mutant is around 3 μM, while WT is around 10 μM. Yet as the bulk of the mutated residue decreases, the ΔE_a increases significantly. In addition, the kinetic isotope effects decrease much more sharply with temperature than for WT. Again, the magnitude of this increased temperature dependence appears to vary inversely with the bulk of the side chain at I553. From these features, and a decline in the magnitude of the pre-exponential factor, the authors argue that dynamics play an important role in encouraging the tunneling reaction.

Well, you know how I love long-range dynamic effects in proteins. But I don't quite buy it here. An argument based on negatives is never very satisfying in any case, and here we have a lot of questions. For one thing, to say that the structure hasn't changed significantly seems overly simplistic to me. The lowest-energy structure may not have been seriously deformed, but absent crystal packing and with a little extra kT around, the average structure might be different. Additionally, these structures were obtained in the absence of ligand. Even though the energetics of binding do not appear to differ significantly for these mutants, the structure of the enzyme or ligand may have changed in the bound state. Even if I accept that the existing structures argue for a completely identical binding site in the free state, and this could realistically be debated, that's no guarantee that the same is true of the bound state.

I'm not denying that the evidence is strongly suggestive, and getting direct data may be difficult given the size of the protein. However, almost all of these side chains are methylated. That means that perdeuteration of the protein in concert with specific side-chain labeling could be especially fruitful in directly observing the dynamics of the pocket during catalysis. It should also in principle be possible to label other side chains in the region to determine how mutations affect the whole area. It is likely that the substrate can also be labeled, possibly with a nucleus or tag that is poorly relaxed. T₂ is likely to be a major challenge here, but not necessarily an insurmountable one, especially since the enzyme appears to be folded and active up to around 50 °C. The presence of the iron is certainly a complicating factor; however, the dynamics should be the same whether catalysis is occurring or not, so possibly it could be substituted with a non-paramagnetic metal. It wouldn't be the easiest project in the world, but it should be doable.

I think these results are intriguing, and I'd love to hear Warshel's take on them. Nonetheless, I feel I have to reserve judgment at least until dynamic changes in the pocket that seem to correlate with the kinetic observations are directly demonstrated by NMR or some other method.

Meyer, M.P., Tomchick, D.R., Klinman, J.P. (2008). Enzyme structure and dynamics affect hydrogen tunneling: The impact of a remote side chain (I553) in soybean lipoxygenase-1. Proceedings of the National Academy of Sciences, 105(4), 1146-1151. DOI: 10.1073/pnas.0710643105 - OPEN ACCESS ARTICLE

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A Chilling Dilemma

Back when I was at Chapel Hill, a fellow student asked my help in figuring out a strange phenomenon he observed with one of his proteins. It seemed that the protein was perfectly soluble at room temperature, but precipitated when it was cooled. Because proteins typically denature at higher, rather than lower, temperatures, he was mystified. Mostly out of luck, the first question I asked him proved to be the right one: "What buffer are you using?" As it happened, he was using Tris buffer, a very common buffer in the biosciences, but also one that changes pH significantly upon cooling. It was not temperature that was denaturing his protein, but rather a change in the alkalinity of the solution it was dissolved in. The change of buffer pH with temperature is one of the most subtle and least-discussed issues in biochemistry, but as in my friend's case, can often produce confusing effects that significantly complicate research. An article by scientists from the University of Illinois online now at Chemical Communications (click link for open access), however, aims to resolve this problem by producing a temperature-independent pH (TIP) buffer for biological applications.

The approach they employed was actually very simple: rather than search the ends of the earth for a buffering agent that would maintain its pH across a huge temperature spread, they just found two that had opposite reactions to chilling and mixed them together. The buffer system they ended up with contained 60% HEPES and 40% potassium phosphate and experienced a pH change of less than 0.07 ± 0.1 over a temperature range from 25 °C to -180 °C. Readers who have encountered this subject before will recognize that HEPES and K₂HPO₄ are already fairly pH-stable over most of the laboratory temperature range, but as the paper's first figure shows, the TIP maintains these benefits down to very low temperature. A colorimetric assay was used to track the pH to very low temperatures.

So, problem solved, right? Well, not exactly. This finding is very nice as far as it goes, and has some promise for labs that do a lot of work that involves freezing proteins—structural biology labs come to mind. But while it would be nice to be able to select buffers on the basis of pH characteristics alone, that's rarely feasible. Not all buffers work with all proteins. In some cases, this can be rationalized: proteins that bind ATP or nucleic acids often bind or interact with phosphate buffers, resulting in poor solubility or aberrant structural dynamics. In other cases the observations are harder to understand—it seems that some proteins just "don't like" some buffers. So long as you're dealing with very dilute proteins, this is not usually an issue, though the biochemistry is sometimes deranged as a result. However, when high protein concentrations are employed—again, this is characteristic of structural biology—incompatible buffer-protein combinations tend to result in crashed protein.

The good news is that, with the exception of proteins that normally interact with phosphate moieties, both of these buffers are fairly well tolerated by a wide spectrum of systems. So long as a nearly-neutral pH is desired, this buffer combination should be useful. Researchers wishing to safely put pH-sensitive small molecules into long-term cold storage are also likely to find this buffer a boon. Moreover, because the buffer components are common and inexpensive almost any lab will be able to use this approach.

Sieracki, N. A., Hwang, H-J., Lee, M.K., Garner, D.K. and Lu, Y. "A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures." Chem. Commun. 2008 DOI 10.1039/b714446f

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Allosteric inhibition in medicine

If you wanted to keep someone from driving off in their car, how would you do it? There are some obvious answers: you could put some kind of blockage in front of and behind it, or you could slash the tires, or you could damage the engine, or you could empty out the gas tank. All of these approaches directly interfere with the mechanisms that allow the car to move. Alternately, you could take a more indirect approach: you could break the doors so they don't open, or remove the steering wheel, or take out the brake and accelerator pedals, or remove the stickshift. None of these latter approaches make it impossible for the car to move — the engine and wheels still function — but by removing the means that allow human beings to control the car these approaches still manage to ensure that the vehicle cannot be driven.

The traditional approach in structure-based drug design is to attempt to block the active site of a protein or important interacting sites, analogous to the "direct" approaches I outlined above. By adding a lump of stuff that obstructs enzymatic activity, ion transport, or (more recently) binding interactions these drugs attempt to interfere directly with the biochemistry of the target protein. This approach has been pretty successful, and certainly no reasonable person would want to depart from it, but there are some weaknesses here. For one thing, many diseases don't originate from enzymes or ion channels, and developing drugs that obstruct binding interactions can be pretty tricky.

Imagine your disease arises in the following system. You have some kind of receptor (R in my little cartoon over to the side there) that can bind a ligand (L). Binding of L activates R in some way so that it can bind to a partner (P) and this causes some gene to be activated. This cartoon roughly represents the way in which hormone receptors (like for estrogen or testosterone) function. Now, suppose something has occurred to derange this system: perhaps too much of L is being made, or R and P have been aberrantly expressed in some tissue where the gene they activate is toxic or causes inappropriate cell proliferation.

If we come at this by the conventional approach of targeting the L-binding pocket, we have a problem, because any drug that has an affinity for R high enough to displace the natural ligand is likely to produce the same or similar conformational changes that cause P to bind. We could, of course, try to develop a drug that interferes with P's binding directly, but this approach has its own problems. For one thing, these binding surfaces are often quite large and highly structured, features that are often difficult to replicate with a small molecule. You can create a drug that inserts itself into the site somehow and gets in the way of P binding, but it's not always possible to design such an agent that specifically hits only your protein. A peptide mimic of R might work, but these are also difficult to deliver.

But there's another strategy that might work, too. Rather than try to block R from binding L or P directly, what if we could cause some other change in R that prevents P binding whether or not L is around? Just like removing the accelerator or stickshift from the car of my example, this doesn't directly block either function of the molecule. R can still bind L, and the binding surface P interacts with is still present. However, our drug (D) has caused a new change in R's conformation that prevents the binding of P. A simplified cartoon is at left.

This is precisely what seems to have happened in a study on the androgen receptor (AR) appearing the October 9 issue of PNAS and reported by Eva Estébanez-Perpiña and coworkers from UCSF and St. Jude's. Interestingly, this group of researchers was initially seeking to produce a direct inhibitor — in this case, a drug that would bind to the part of AR that interacts with its coregulators. This interaction is therapeutically interesting because aberrant AR activity plays a role in prostate cancer, among other things. Several promising compounds were found that produced substantial inhibition of AR activity at relatively low concentrations. The surprise was that only some of these preferred to bind the targeted site (AF-2). Instead, a number of them were found to locate preferentially to a previously unsuspected site at the top of the molecule, now called binding-function 3 (BF-3), which you can see in the figure I shamelessly stole on the right here.

The reason that this appears to work is that binding to BF-3 changes the structure of AF-2, via an allosteric interaction. Rather than directly getting in the way of the essential reaction, the drug acts at a distance to disrupt the binding surface. Although much still needs to be done to refine these lead compounds into usable drugs, this is a significant demonstration that such an approach has therapeutic potential. In this case, the discovery of allosteric inhibitors was made by accident, but as our understanding of protein structure and allostery improves, expect to see more efforts to approach drug design in this way.

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