Bailing into the lifeboat

A few weeks back, when I posted about the necessity of raising taxes and cutting spending in order to reign in the national debt, I did not anticipate that the market would force us to take such an enormous step towards insolvency. The bailout proposed by the Bush administration would cost us $700 billion, but many predict that its actual cost, after Congress adds its own trimmings and attempts to include some relief for homeowners in danger of default, will reach $1 trillion or more of money that we don't have. The irony of paying for bad debt with more debt might inspire a grim sort of humor if we weren't in such a big hole already. The plan as initially proposed is completely outrageous, but I have little doubt that Congress will find some way to make it worse.

The fundamental problem is that a mortgage-backed security is not a mortgage. The holder of a mortgage has the right to foreclose on the property (thus recovering real assets) if the loan is not repaid, but the holder of a MBS does not appear to necessarily have this right. The security spreads the risk from a pool of mortgages among a pool of investors; as such, no single investor can be said to "own" a particular mortgage (except in the case that he owns all the securities from a given pool). It's not clear whether the owners of MBS own anything other than debt; it is possible that they do not own the underlying loans. And these are the simplest vehicles assembled from residential mortgages... the real value of more exotic derivatives may be impossible to assess. This has serious implications for the proposed bailout, because there is a very real chance that we taxpayers will end up paying billions of dollars for smoke and mirrors. Before we release one penny from the Treasury for this rescue effort we must ensure that what we purchase with our money will give us a right to the underlying property, as well as the authority to modify the mortgages so as to diminish the default rate. We must not be left holding a bag of air.

The uncertain relationship between the securities and the actual mortgages underscores the unseemly nature of the whole affair. People who did not have the wherewithal to own homes got mortages from unscrupulous lenders who should never have given them out. These mortgages were packaged into vehicles that were treated like gold by credit raters, and then purchased by investors who probably should have known better. Only a fool could have imagined that the housing boom would continue indefinitely. The push by credit providers to make bankruptcy declarations more difficult for individuals had the unexpected side effect of increasing defaults. Holding negative equity on their homes in a plunging market, homeowners simply turned off the lights and walked out. The invisible hand failed to reign in the cascade of short-sightedness, stupidity, and outright malfeasance, and the credit market landed on its head with an audible crunch.

This would be bad enough on its own, but it set off a chain reaction leading to ever more violent flailing on the part of the Treasury department and the market players themselves. Because the securities had been insured, AIG took a hit and needed billions of dollars of government money just to die quietly. The failure of Lehman Brothers left money market funds holding worthless paper; the Reserve Primary Fund broke the buck and skittish investors started to flee. With all business in danger of grinding to a halt because of the shortage, the Treasury insured these investments with the Exchange Stabilization Fund. Because this insurance is not capped, small banks are now worried that panicked customers may move all assets in excess of $100,000 into the money market, leaving them short on cash.

Keating Five member John McCain, long a friend to unscrupulous financiers and enemy to the kinds of regulatory oversight that might have prevented this crisis, has been difficult to pin down on this issue, in large part because his position changes every time the sun comes up. His initial position, that we should stop bailing out financial giants, is understandable and at least has the virtue of being consistent with his free-market philosophy. His later attitude, an acceptance of the reality that these companies must be bailed out in order to protect the investors who acted in good faith, was more realistic. Exposure to the toxic mortgage-based securities put the other assets of these companies at risk, and it would be unconscionable to destroy the investments of good actors as punishment for the deeds of bad actors who had already escaped on golden parachutes.

(In McCain's defense, at least he said something, even if it was insane, and Joe Biden's response tracked a similar trajectory. As for the other presidential candidate, Barack Obama couldn't manage anything better than "I'll get back to you on that." Who will you choose in November: the madman or the slacker?)

Bailing out rich people stinks, but it stinks more when we don't really have any money to do it with. Section 10 of the proposed legislation increases our national debt limit for the fiscal year to $11,315,000,000,000. Given the authority to hit that ceiling, I have no doubt that the Treasury department will do so, meaning that our interest outlay in the next budget will be even higher than previously anticipated. Every new program you've heard politicians mention during this election cycle has just evaporated. The cost of a bailout is a credit crunch on the government, at the worst possible time. I've already proposed the solution to the problem: we must increase income and cut outlays. Otherwise, we're just bailing water from the yacht into the lifeboat. Specifically, the wealthiest Americans must be willing to pay higher taxes, because it is they that benefit most from the financial institutions the rest of us will be breaking the bank to rescue, and it was their exploitation of those markets that got us into this mess.

Of course, I haven't yet gotten to the most malodorous part of the whole proposal. It's bad that we don't know whether what we're buying will be worth anything, and it's worse that we'll significantly increase the national debt to do so. But the really despicable part of the legislation is this:

Sec. 8. Review.

Decisions by the Secretary pursuant to the authority of this Act are non-reviewable and committed to agency discretion, and may not be reviewed by any court of law or any administrative agency.

You know, at least with the absurdly-named Patriot Act these Republican pricks had some kind of flimsy excuse for their totalitarian actions. This is sticking a thumb in the eye of Democracy just to show you can. It was a lack of transparency and honesty that got us into this whole mess; we cannot get out of it by spending $700,000,000,000 at the sole discretion of faceless bureaucrats from an abominably opaque and secretive administration. The estimated cost of the Iraq War to date is around $582 billion, a massive number but still less than the amount proposed in this legislation. It defies reason and sense to insulate the choices made with this massive amount of money from oversight and accountability. The very request for opacity suggests that the whole operation is being undertaken in bad faith.

Bernanke and Paulson continue to insist that Congress must act immediately. Nothing in their past behavior, however, suggests that they possess the competence to make this analysis, or the honesty to accurately convey their analysis to the media or to the legislature. Clearly, something must be done, but handing out $700 billion to the same nitwits that got us into this mess, without anything even resembling appropriate oversight, is more likely to bring economic disaster than salvation.

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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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Upside down

As a very important source of strength and security, cherish public credit. One method of preserving it is, to use it as sparingly as possible; avoiding occasions of expense by cultivating peace, but remembering also that timely disbursements to prepare for danger frequently prevent much greater disbursements to repel it; avoiding likewise the accumulation of debt, not only by shunning occasions of expense, but by vigorous exertions in time of peace to discharge the debts, which unavoidable wars may have occasioned, not ungenerously throwing upon posterity the burthen, which we ourselves ought to bear. The execution of these maxims belongs to your representatives, but it is necessary that public opinion should cooperate. To facilitate to them the performance of their duty, it is essential that you should practically bear in mind, that towards the payment of debts there must be Revenue; that to have Revenue there must be taxes; that no taxes can be devised, which are not more or less inconvenient and unpleasant; that the intrinsic embarrassment, inseparable from the selection of the proper objects (which is always a choice of difficulties), ought to be a decisive motive for a candid construction of the conduct of the government in making it, and for a spirit of acquiescence in the measures for obtaining revenue, which the public exigencies may at any time dictate.

—George Washington, Farewell Address

The next President and Congress of the United States must raise taxes and cut spending.

The last time I checked, the national debt was about $9.6 trillion, but it's probably higher now. The interest on this debt presently amounts to nearly 10% of the federal budget, which admittedly is less than half of we spend on defense, but still easily exceeds $200,000,000,000. According to the Congressional Budget Office, the interest outlay in 2007 could have covered the entire federal expenditure on supplemental security income, child tax credits, unemployment, food stamps, family support, child nutrition, and foster care, with almost enough left over to pay for veteran's benefits. Of course, the magnitude of the debt (and the associated interest payments) will only increase when Medicare and Social Security payments start to exceed revenues. The federal government has run a deficit every year since 2001, and none of the people running for office now have done enough to stop it. Nor do they propose to do enough to stop it.

Instead, our candidates propose hosts of new projects, proposing no financing beyond the sunshine and rainbows that adorn their professionally-coiffed speeches. They shoo away the petty earmarks while the great consumers — Medicare, Social Security, Defense — gnaw the budget to the bone, unmolested in their gluttony. Most candidates who are willing to tax are unwilling to stop spending; most candidates who are willing to cut expenditures also want to lower taxes. Either way is insane, but many candidates want the whole pie: more spending, less taxes, damn the torpedoes, full speed ahead!

It has become almost obligatory, especially among those seeking office for the first time, to blame the present excesses on fat cats in the capital, but simply pointing the finger at Washington doesn't cut it. Irresponsible big spenders do not reach office by magic; they get there because we elect them. This is not a problem of other people in other districts who have bad judgment and choose lousy representatives. This is your problem in your district, and it is a problem because we have failed to do as George Washington asked. A man who tells you that our budget problems can be solved without any pain is either a fool, or a liar who thinks that you are a fool. But when a candidate gets up and acknowledges that taxes must be raised, that favored spending must be restrained, he is hated by an electorate that ought to applaud him for his honesty.

So the politicians lie. They cut taxes and spend more. They raise taxes and outspend that. And all along we grumble and complain about "Washington insiders" and "Beltway bandits". Well, throw the bums out! That's in your power, isn't it? If you're reading this post you have access to the internet. Find out how your congressman voted during his term. Read the text of the bills he voted on. Find out what questions he asked in committee and what speeches he made on the floor. Dig up the things that interest you, no matter how obscure. The behavior of state legislators and other officials is often more difficult to track, but these records exist. You as a voter have a duty to find and evaluate them, because terribly few politicians are honest about their record or intentions. Even if they are inclined to tell the truth, they dare not speak it because we voters are all too happy to punish harsh truths and reward pleasant lies.

Paying down the national debt is a personal obligation. We owe it to our children and grandchildren, our nieces and nephews, not to leave them a country upside down on its loans. More than that, however, it is a patriotic obligation. We owe it to our parents and grandparents, our soldiers and our founders, not to let the country they worked so diligently to build and preserve crumble due to debt and dissipation.

The next President and Congress of the United States must raise taxes and cut spending. Any candidate for any national office who refuses to acknowledge this truth does not deserve your vote.

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Love your Playstation with Odin Sphere

The big news this week belongs to the XBox 360, of course. As the internets knew, like, three weeks ago, all XBox models have a price drop coming on Friday, with the Arcade hardware (note: do not buy the Arcade hardware) dropping to $199, less than the Wii. The standard hardware will come in at $299, a price that is sure to help with sales for the upcoming holiday season. But, maybe you don't have $300 for a next-gen console, or even $60 for a next-gen game. Maybe all you've got is $20 burning a hole in your pocket and a PS2 that needs a good workout. If so, then you just might get your money's worth from this week's "Greatest Hits" re-release of Odin Sphere, a hybrid side-scrolling brawler/RPG from Atlus. That's an excellent deal on one of the most beautiful and interesting games of the last hardware generation.

I've written about Odin Sphere on this page before, discussing both its effective use of the cycle motif (spoilers in that post) and its rather less effective coding. I rarely make a case for a sale on this blog, as I prefer to simply discuss what I feel is going on in terms of story and imagery, but I make an exception in this case because Odin Sphere is, well, exceptional. The quality of the visual presentation cannot be overstated — this is one of those cases in which the game in motion looks better than the screenshots. The soundtrack by Basiscape is just fantastic. This presentation frames a deep and moving story that harkens back to Norse mythology and Wagnerian epics without quoting them so directly that it seems simply recycled.

That said, Odin Sphere is also deeply flawed, beset with terrible framerate reduction in some boss fights and long loading times. The apparent absence of cancels from the combat system exacerbates the frustration arising from the framerate problem. The inventory should have been larger; the recipe systems for food and alchemy should have been better refined. Some of these errors amount to mere niggling annoyances, but others can be quite exasperating. I can see why someone might be dubious about investing $40 or more into a game that puts those kinds of obstacles in the way of its glorious art. And, of course, the dreaded JRPG label probably scared away a few folks too.

A flawed diamond, however, is still a diamond. Odin Sphere's problems mean it will never sit atop the list of best games of all time. It is, however, a compelling story presented with largely enjoyable gameplay and stunning visuals and music. Even if you don't usually like RPGs, that's a combination that's worth 20 bucks.

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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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Guided by the (blue) light

The ability to sense and respond to magnetic fields is a fundamental aspect of behavior in many animals. While migratory birds famously use the earth's magnetic field to navigate during, magnetic field responses occur in all manner of animals, from eels to invertebrates. Even the lowly fruit fly, best known as a reminder that you really should have taken the garbage out two days ago, can react to magnetism. While various explanations have been put forward in different species, magnetosensitivity remains fairly mysterious. In this week's Nature, researchers from the University of Massachusetts Medical School show that the blue-light photoreceptor cryptochrome plays an essential role in allowing fruit flies to detect magnetic fields.

Cryptochrome (or Cry) inherited the ability to receive blue light along with its photolyase domain, which is homologous to a prokaryotic, light-dependent DNA repair protein. Cry proteins, which are present in all animals, do not perform any DNA repair work, but instead play a role in regulating the circadian rhythm. While it is not clear in all cases whether Cry's ability to absorb blue light is biologically significant in clock regulation, it is known that fruit flies (Drosophila melanogaster) use Cry to synchronize their circadian clocks. Previous experiments had suggested that the ability of fruit flies to detect magnetic fields was somehow related to photoreception, and that short wavelengths (like those sensed by Cry) had different effects from longer ones.

Gegear et al. devised a relatively simple experiment to test the importance of Cry in Drosophila magnetosensing. They placed a T-junction in a box, with a magnetic coil on one side and a non-magnetic coil on the other. They released flies into the junction, with (trained) or without (naive) performing an earlier run where the magnetic field was associated with a sucrose reward. They shined a light into the box and used filters to investigate the role of specific wavelengths.

They discovered that several strains of Drosophila could be trained to go to the magnetic field, although the degree of preference and the nature of the naive response differed substantially between strains. Gegear et al. chose the strain that showed the greatest response in full-spectrum light (and displayed a tendency to avoid the magnetic field in the naive state) to perform the filter experiment. Cutting off all wavelengths of light shorter than 500 nm abolished both the naive and trained responses to the magnetic field in these flies, as did filtering out all wavelengths shorter than 420 nm. If only wavelengths shorter than 400 nm were cut off, some of the trained and naive response returned. Simply dimming the light was not enough to replicate the effect of filtering. These experiments indicate that magnetic sensitivity in these flies requires light in the blue to ultraviolet range.

In order to prove that cryptochrome specifically is necessary for this magnetic sensitivity, Gegear et al. took advantage of our tremendous knowledge of fly genetic manipulation to create mutant flies that did not have a functional Cry gene. No matter what wavelengths of light were used in the T-junction experiment, these flies did not respond to the magnetic field. Crossing these Cry-null mutants with normal flies restored magnetosensitivity. The authors also performed experiments to show that the circadian rhythm was not itself essential to magnetic response in the flies.

Because this is a genetic experiment, it cannot address the question of whether Cry is both the blue-light photoreceptor and the magnetosensor. Going just on what we have in this paper, it is also possible that Cry acts upstream of another magnetosensor protein or is part of its downstream signaling pathway. However, in light of research that shows the flavin photoreception in other cryptochromes induces the formation of magnetically-sensitive radicals, some of which I discussed last year, it certainly seems possible that Drosophila cryptochrome does the whole job itself. As I mentioned in the case of the previous article, though, there is not yet any understanding of a mechanism by which information about magnetic field could be transduced from Cry radicals into the nervous system.

Dorosophila Cry differs from other plant and animal Cry proteins in significant ways, so it's unclear whether these results have any relevance for other organisms. However, the finding that Cry is essential to Drosophila magnetosensitivity suggests at least the possibility of parallel systems in migratory birds and other species that use magnetic fields.

Robert J. Gegear, Amy Casselman, Scott Waddell, Steven M. Reppert (2008). Cryptochrome mediates light-dependent magnetosensitivity in Drosophila Nature, 454 (7207), 1014-1018 DOI: 10.1038/nature07183

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How to help an enzyme crack cocaine

In addition to the adverse consequences of addiction and the inconvenience of serving several years of jail time for possessing it, cocaine can cause a fatal overdose. Although this condition can be treated, no therapy presently exists that attacks the overdose by removing cocaine from the bloodstream. One possible approach to eliminating cocaine from a patient would be to accelerate the process by which it is degraded. Unfortunately, the enzymes that perform this activity in the human body are not very efficient. In an upcoming article in the Journal of the American Chemical Society, however, a group from the University of Kentucky (assisted by researchers at the University of Michigan) have remodeled the active site of butyrylcholinesterase (BChE) to achieve a 2000-fold increase in rate. This raises the possibility of producing therapeutic enzymes as a treatment for cocaine overdose.

A cocaine overdose typically results in an elevated pulse rate, seizures, and hyperthermia, among other possibilities. The usual course of treatment involves addressing the symptoms — diazepam to reduce the heart rate, cooling protocols to address hyperthermia. These steps are proven to work, but they don't address the core problem: there's still a lot of cocaine floating around in the bloodstream. Treating with sedatives amounts to using one giant truck to stop another giant truck... both trucks will probably stop, but there might be a lot of collateral damage. Instead, it would be advantageous to either block the receptors that cocaine binds, or clear cocaine from the bloodstream somehow.

Plasma butylcholinesterase does most of the work in metabolizing cocaine, by cleaving it into two products that no longer exert the same pharmacological effects. If BChE was a highly efficient enzyme it's unlikely that people would experience cocaine overdoses at all, but it breaks down the main form of cocaine quite slowly, with a catalytic rate (k_cat) of 4.1 /min, resulting in a very long half-life for this substrate. The chemical mechanism of BChE (Figure 1) will be familiar to anyone who has taken biochemistry, being basically the same as a serine protease. Instead of a peptide bond, however, it is the ester linkage of cocaine that undergoes nucleophilic attack from an activated serine, while hydrogen bonds stabilize the evolving negative charge in an oxyanion hole.

Previous efforts to optimize the activity of BChE by mutation focused on eliminating steric clashes, but Zheng et al. noted that the hydrogen bond lengths in the oxyanion hole were not optimal for stabilizing the putative transition state. They therefore decided to focus their efforts on improving the energetics of this region. To do so, they used combined quantum mechanics/ molecular mechanics (QM/MM) simulations to determine the energy barriers in simulated reaction coordinates for a number of different mutants. This has the advantage of screening potential mutants for a specific effect, which may be quicker than wet lab work, but it requires the researcher to know the catalytic mechanism and to define a region of interest in advance.

By working through a series of mutations, Zheng et al. arrived at one multiple mutant of BChE that had favorable interaction energy for every residue in the oxyanion hole. When they generated this mutant in the lab, they found that it had a vastly increased catalytic rate towards cocaine, with k_cat now about 5700 /min. Based on these in vitro results they decided to test the mutant BChE in vivo using mice. They found that injecting mice with 30 µg of BChE protected them from seizure and death due to cocaine overdose. While the n for this experiment is small, and the BChE was injected prior to cocaine exposure rather than after, these results suggest that the mutant BChE has potential as a therapy for cocaine overdose in humans.

Obviously, further improvement would be needed before these protective effects could be realistically equaled in humans. To match the dose used in this experiment, a 180-pound man would need to be injected with 82 mg of the protein, which is a rather large amount. However, if used in conjunction with existing treatments, the required dose of BChE may be lower. If not, then translating these results into a useful therapy will require either further catalytic optimization or an enormous production effort. A significant amount of additional clinical research is required before this or any other mutant of BChE is introduced as a therapy for overdose or addiction. Nonetheless, these results illustrate the promise of enzyme optimization and design as a tool for medicine in the future.

Fang Zheng, Wenchao Yang, Mei-Chuan Ko, Junjun Liu, Hoon Cho, Daquan Gao, Min Tong, Hsin-Hsiung Tai, James H. Woods, Chang-Guo Zhan (2008). Most Efficient Cocaine Hydrolase Designed by Virtual Screening of Transition States Journal of the American Chemical Society DOI: 10.1021/ja803646t

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Two great mechanisms that go great together

The watery interior of a cell is separated from the watery exterior of a cell by a thin double layer of lipids called the plasma membrane. The oily interior of this membrane prevents water and charged molecules from escaping the cell, while allowing hydrophobic (oil-like) molecules through. This system has many significant advantages, but cells frequently need to move charged atoms (ions) across the membrane. This job is primarily performed by two kinds of protiens: channels that create specialized tunnels through the membrane, and transporters that mechanically transfer ions across the membrane. These are distinct activities, usually carried out by different families of proteins. Recent results in the ClC family of membrane proteins, however, have demonstrated that these activities are not as distant from each other as it might seem.

Ion channels basically work like tiny pipes that stick through a cell membrane. They have interior pores that are full of water, and usually possess some form of selection mechanism that lets only a particular kind of ion through. Although they can be gated — opened or closed by particular voltage states or molecular signals — they can only move ions with an electrochemical gradient. That is to say, they can only allow their particular ions to move across the membranes in a way favored by both concentration and voltage. By contrast, transporters physically translocate ions without using a watery pore. This allows them to move ions against a concentration gradient as long as they have an energy source, such as another ion's concentration gradient.

The figure cave drawing at right shows a simplified situation. A membrane divides two compartments, one of which has a high concentration of negative ions (red), while the other has a high concentration of positive ions (blue). The selective channel (C) can only allow negative ions to move from the high to low concentration in this situation, because the concentration and electrical gradients oppose a movement in the opposite direction. The transporter (T), on the other hand, can push negative ions out into the area where they have a high concentration. It does this by simultaneously transporting a positive ion from high to low concentration; it uses the favorable energetics of this transport event to power the unfavorable one. Of course, there are many different kinds of transporters -- the example here is a symporter, but there are also antiporters or exchangers.

Aside from dealing in ions, these two kinds of activity might seem to have little in common, and in fact most ion channels are not very closely related to ion transporters. However, the ClC family of chloride channels is unusual in that it also includes several members that are transporters. Because all these proteins are presumed to have similar structures in the membrane, there is considerable interest in understanding the key differences that separate ClC channels from ClC transporters. In the case of these proteins, it seems that the line between transporter and channel is easily blurred.

You got transporter in my channel!

The ClC-0 protein from the electric ray Torpedo marmorata has always been classified as a gated channel, but an odd one. Unlike many channels, it has two pores in its active configuration. These pores are closed off or gated by two processes. The fast gating occurs on the millisecond timescale and opens and closes the two pores independently of one another. The slow gating occurs on the timescale of seconds and opens and closes both pores.

The odd thing is that there is a thermodynamic imbalance in this system. All things being equal, we expect that if we track the number of pores that are open over time, we should see the pattern 1 → 0 → 2 → 1 (called J₊) with the same frequency that we see the pattern 1 → 2 → 0 → 1 (J_-). Instead, researchers have found that J₊ is observed more frequently than J_-. Originally it was thought that the electrochemical gradient of chloride controlled this asymmetry, but this model didn't quantitatively match the observations. Now Lísal and Maduke have shown that the asymmetry arises from the proton electrochemical gradient, and that this is a molecular vestige of ClC antiporter function (1).

The evidence for this comes from an experiment in which the chloride electrochemical gradient was held constant while the proton electrochemical gradient was changed. If the chloride gradient controls the asymmetry, we should see no differences in the J₊/J_- ratio during the experiment. Instead, it was observed that switching the direction of the proton gradient changed the behavior of the channel gating from almost exclusively J₊ to an even mixture of J₊ and J_-. Further experiments demonstrated that the J₊/J_- ratio was proportional to the proton electrochemical gradient, and that it leveled off at 1 when the direction of the gradient was changed to favor movement of protons from inside the cell to outside the cell. This suggests that the gating mechanism is unidirectional.

Because the proton gradient provides energy to produce gating asymmetry, CLC-0 must be a proton transporter as well as a chloride channel. The authors suggest that this reflects a vestigial antiporter activity (similar to several existing members of the family) that has been repurposed as a regulatory mechanism for channel gating. Although channels have very different thermodynamics from transporters, it appears that a single protein can have characteristics of both.

You got channel in my transporter!

But how does an antiporter transform into a channel? Because the structures of these different types of proteins tend to be quite dissimilar, it seems unlikely that the transition between these mechanisms could be accomplished with just a few point mutations. However, sequence homology in a family of proteins usually implies structural homology (with some exceptions), so the ClC channels probably evolved from antiporters in this fashion. This could happen by the destruction of gating mechanisms in the transporter.

In the Cl^-/H⁺ antiporter CLC-ec1, a central binding site for chloride is blocked by two gates. One side is blocked off by Glu148, and the other is putatively formed by the interaction of Tyr445 and Ser107. In principle, removing these gates could give rise to a watery passage through the membrane, and high transport rates similar to those of ion channels. Jayaram et al. therefore performed a series of mutations at these sites and measured the effect on transport rates (2).

From previous work it was known that altering these residues uncoupled chloride transport from proton transport, which is one step towards becoming a channel. Here, Jayaram et al. find that mutating Tyr445 to a smaller side chain does not have a significant effect on chloride transport rates. Mutating Glu148 to smaller residues actually decreases the chloride transport rates. Mutating both residues, however, leads to a 100-fold increase in chloride transport over the wild-type protein. The fastest mutant, moving chloride ions at a rate of more than 35,000 /s, is not quite as fast as a channel. Still, the acceleration is significant.

Jayaram et al. found that increasing the size of the substituted side chain at either site decreased the rate. This is what you would expect for a simple case of larger side chains leading to more constriction of a channel. In order to confirm that there was such a channel, they crystallized one of the mutants and analyzed it to determine whether there was a continuous pathway that water could permeate. Below, you can see a figure I shamelessly stole from their paper, with the WT protein on the left and the E148A/Y445A mutant on the right. The red dots represent a surface made with a 1.4 Å probe designed to mimic water. As you can see, the probe can go all the way through the A/A mutant, but is stopped by the intracellular gate in WT. The double mutant is a tight, but genuine channel.



Because these residues are conserved in both channel and antiporter members of the ClC family, mutations like these are not likely to be the means by which one kind of protein evolves from the other. Nonetheless, they establish that the crystal structure of CLC-ec1 is likely to be a good model for occluded states of gated ClC channels. Moreover, the ease with which a specialized ClC transporter is made into a channel suggests that a progenitor protein could have switched from antiporter to channel in just a few mutations.

The thermodynamics of ion translocation by channels and transporters are quite different, so it was a surprise to discover that the ClC family contained both kinds of activities. These recent papers show that the entanglement is even closer than previously thought. The ClC channel CLC-0 still retains vestigial proton transport activity, and the ClC antiporter CLC-ec1 is only a few mutations away from becoming a channel itself. These findings suggest that substantial changes in operating thermodynamics may result from small evolutionary steps, and point to a shared antiporter past for members of the ClC family.

1. Jiří Lísal, Merritt Maduke (2008). The ClC-0 chloride channel is a 'broken' Cl−/H+ antiporter Nature structural & molecular biology, 15 (8), 805-810 DOI: 10.1038/nsmb.1466

2. H. Jayaram, A. Accardi, F. Wu, C. Williams, C. Miller (2008). Ion permeation through a Cl--selective channel designed from a CLC Cl-/H+ exchanger Proceedings of the National Academy of Sciences, 105 (32), 11194-11199 DOI: 10.1073/pnas.0804503105
Disclaimer: I work on the same floor as all these guys.

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How media resemble real life in your head

How does the human brain react to the communication of emotion? Does the observation or imagination of emotions have anything in common with the personal experience of them? It is possible that the brain uses a setup in which seeing a person experience an emotion, imagining that emotion, and feeling that same emotion all use completely independent circuitry. Yet since all of these experiences make references to the same emotional state, it is also reasonable to think that some of the pathways are shared. In a recent article from PLoS ONE, a team of researchers uses functional Magnetic Resonance Imaging (fMRI) to determine similarities and differences in the patterns of brain activation following various means of communicating disgust. PLoS ONE is open access, so go ahead and open the article up in another window.

First, a word about fMRI, for those unfamiliar with it. As the name would suggest, fMRI is an elaboration of the standard MRI techniques used image the interior of your body without the use of potentially harmful radioactivity. Neuronal activity in the brain causes a local depletion of oxygen from the blood, followed by a localized increase in blood flow. Because the magnetic properties of iron in the blood change with its oxygenation state, it is possible to detect these hemodynamics using magnetic resonance imaging. Thus, fMRI is able to indirectly detect neural activity, although the fMRI signal lags behind activity by a few seconds. A given fMRI signal also encompasses a large number of individual neurons and therefore can only serve as a rough map to where things are happening in the brain. These temporal and spatial limitations limit the conclusions that can be drawn reliably from fMRI, but the observed correlations can provide valuable insights.

Jabbi et al. used fMRI to map the neural response of subjects to various encounters with disgust. Previous research had shown that a particular region of the brain (the IFO) showed increased activity when subjects either tasted something disgusting, or viewed a short clip of someone else tasting something disgusting. For this study, Jabbi et al. had participants read short scripts (samples can be found in the supplementary materials) intended to make the reader imagine being disgusted, pleased, or not feeling anything. They found that reading disgusting passages induced a neural response in this region of interest, just as it had for the cases of tasting or observing disgust.

While this may seem completely unsurprising, it bears some consideration. The experience of personal disgust differs significantly from the experience of observing disgust in others. Similarly, imagining or reading about disgust creates a very different subjective experience than, say, drinking quinine. Given that these are all quite different feelings, it is somewhat surprising that a single area is activated by all three.

Of course, there is a fine line to consider here -- the passages meant to make the subjects imagine disgust may have actually disgusted them. The paragraphs that the authors make available in the supplementary materials are written in second person and involve things like accidentally ingesting animal waste. Because the subjects are reading passages that ask them to imagine themselves being disgusted, and the passages are themselves disgusting, the act of imagination may be contaminated by an immediate personal experience of disgust. In a more elaborate experiment it might be of value to use passages written in the third person. Additionally, it might be useful to employ passages in which the characters, because of particular phobias or personal experiences, are disgusted by items or actions the reader is likely to find innocuous.

Whether the readers where themselves disgusted or not, the overall response in the brain differed for each of the stimuli, as shown by a map of correlated activity (Figure 2). While the area outside the IFO activated by observation was relatively small, both the disgusting taste and the disgusting scripts produced widespread activity relative to a neutral taste or script. In general there was not much overlap between the networks, except for a small region shared by the imagination and experience groups. The authors propose that the similarities of imagining, observing, and experiencing emotion are due to the common activation of the IFO, while the differences between these are due to the largely distinct networks of correlated activity. Different modes of exposure to disgust may therefore act in complementary, rather than independent, ways.

Additionally, this result appears to be consistent with the view that our recognition of observed disgust and our imagination of disgust rely on an internal simulation of our own feelings of disgust. However, these experiments cannot establish exactly what a particular region of the brain is doing, so this remains an open question.

While this research does not indicate whether these results can be generalized to other emotional states, this finding may interest developers of media that make use of multiple modes of communication, specifically video games. Games often rely on video cutscenes to convey story and emotion, but this approach may be wasting a significant amount of potential. The participatory nature of games makes it possible to approach emotional communication not only through the observational route, but also the experiential route.

Consider the case of Agro's fall in Shadow of the Colossus. Observing the cutscene, and hearing the voice of Wander, the player can understand that Wander feels grief at this event, in much the same way that anyone watching a movie could understand it. Additionally, the emptiness of the game's landscape and the forced collaboration between the player and the Agro AI has helped to create a relationship between the player and the horse. Thus, in observing Agro's fall, the player may feel his own sense of grief at the event, increasing the emotional resonance of the moment.

This suggests a possible, if lengthy, experiment. It would be interesting to compare the fMRI profile of a subjects observing Agro's fall under two conditions: one in which they have actually played the game up to that point, and another in which they have watched the game as a movie, with exploration and battles recorded previously from an expert player's run. Would the first group have activity in both the observational and experiential networks, or would each group activate a different network? What implications might these outcomes have for the development of emotionally fulfilling games?

Of course fMRI studies are not some holy grail that makes everything clear. The work of Jabbi et al. has given us a rough map to where things are happening, but understanding exactly what is happening and how it is happening will require additional experiments and possibly new investigative techniques. Nonetheless, this is an interesting piece of the puzzle, and perhaps some food for thought.

Mbemba Jabbi, Jojanneke Bastiaansen, Christian Keysers (2008). A Common Anterior Insula Representation of Disgust Observation, Experience and Imagination Shows Divergent Functional Connectivity Pathways PLoS ONE, 3 (8) DOI: 10.1371/journal.pone.0002939

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Do conformational changes precede or follow binding?

The binding of a ligand to a protein rarely occurs with the simplicity of a block sliding into an appropriately-shaped hole. Protein and ligand often engage in complementary conformational changes to adapt their shapes to each other. As a result, the structure of a protein bound to its target may differ substantially from the structure of the free protein. Unfortunately, it is virtually impossible to view the binding process in fine structural detail; as a result, most of our knowledge comes from the relatively stable bound and free states. Improving biophysical techniques, however, have brought a change in the way we view some binding events.

Most alterations of conformation during a binding event have historically been interpreted using the induced fit model. In this view, the protein stably maintains the free or "open" structure until it comes into contact with a ligand molecule. This encounter stimulates a conformational change so that the protein adopts the "closed" conformation that tightly holds onto the ligand. Thus, the ligand induces the conformational change necessary to form the bound, closed (BC) structure from the unbound, open (UO) structure, and the intermediate on this path is some kind of bound, open (BO) structure. This model is physically reasonable and has been very successful in interpreting many systems.

However, for the past few decades an increasing amount of evidence has suggested that this is not the whole story. NMR investigations indicated that instead of remaining in a single, well-defined backbone conformation most of the time, many proteins experienced significant changes in their structure while floating free in solution. These results suggested an alternative mechanism of population shift. In this view, the protein actually samples the "closed" conformation (or something very similar) while unbound, and it is this conformation that binds to the ligand. We still go from UO to BC, but now the intermediate is an unbound, closed (UC) structure.

This sounds very arcane, but it is not without functional relevance. Consider, for instance, a protein that is activated by a particular ligand. If we wish to make a drug that binds exclusively to the BC form, then we may experience unforeseen side-effects if our target protein occasionally samples a UC state. It would be useful to have a general idea of what kinds of circumstances are likely to favor a population shift model vs. an induced fit model. That is precisely what Kei-Ichi Okazaki and Shoji Takada aim to provide in an upcoming paper in Proceedings of the National Academy of Sciences (1).

Okazaki and Takada performed a coarse-grained molecular dynamics simulation of glutamine binding protein. In the bound and unbound states they employed a double-well Gō model, a simplified representation of molecular forces, to represent "opening" and "closing". To switch between these states (i.e. to represent binding) they used a Monte Carlo algorithm. This approach has the advantage of being quick and relatively inexpensive from a computational standpoint, but the results must be interpreted cautiously because the physics of the model are greatly simplified. They observe UO ↔ UC and UC ↔ BC events in this system, but they also observe UO ↔ BO and BO ↔ BC events. This suggests that the simulation will be able to make predictions about both population-shift and induced-fit mechanisms.

In order to try to make some predictions about the circumstances in which a particular mechanism is favored, Okazaki and Takada varied the strength and range of the binding interaction. By monitoring whether the simulated system entered the BC state from BO or UC, they could tell whether the system obeyed the induced-fit or population-shift mechanisms, respectively. They find that as either the strength or the range increase, the induced-fit mechanism is increasingly favored (Figure 4). These results make sense. If the protein regularly samples the closed state while unbound, then the amount of energy needed to reach that state is probably small, so it makes sense to see a population-shift mechanism associated with low-energy binding. Similarly, if a ligand is to associate productively with a non-optimal protein conformation, it makes sense that key interactions will be effective at long range.

From these results Okazaki and Takada suggest that the binding of small hydrophobic ligands is generally likely to proceed via population shift, while the binding of large, charged ligands (such as DNA) will likely proceed via induced fit. They acknowledge, however, that the simulation is limited, particularly in its view of conformational change. Unitary transitions in which the whole protein changes its structure simultaneously are probably not the norm, particularly in the case of very large conformational changes. These changes may instead be stepwise or hierarchical. For instance, a protein or complex recognizing multiple features of a DNA strand may proceed by an apparently induced-fit mechanism, even though each individual binding event more closely resembles population-shift behavior.

An additional limitation of this study is that it considers only one protein, but mechanisms of binding and conformational change may be idiosyncratic properties of particular folds. One could consider the behavior of lymphotactin, which displays clear hallmarks of the population-shift mechanism despite binding to macromolecules (heparin and a GPCR) much larger than itself, as a counterpoint to the predictions developed here. Similarly, the population shift of NtrC involves a charged phosphate group likely to have long-range interactions, although this is a post-translational modification and not a strict ligand-binding event. While the authors point to some examples that match their expectations, overall the data are not unanimously in support of their predictions. Still, the general rules laid out here provide a starting point for experimental work.

Despite the limitations of the simulation, it provides a relatively efficient tool for assessing these processes in other proteins. While no simulation can yet replace experimental data, coarse-grained models like this can serve as a means to formulate testable hypotheses about the energetics of protein-ligand systems.

1. Okazaki, K., Takada, S. (2008). Dynamic energy landscape view of coupled binding and protein conformational change: Induced-fit versus population-shift mechanisms. Proceedings of the National Academy of Sciences 105(32) 11182-11187. DOI: 10.1073/pnas.0802524105

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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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