PDZ domains allosterically regulate the bacterial envelope protease DegP

Anyone who reads this blog regularly knows that I have a great deal of interest in the allosteric potential of the PDZ domain, a small protein binding domain that can be found in every branch of the tree of life. In previous posts I've discussed the evidence for allosteric communication within the PDZ domain, as well as evidence for long-range energetic interactions. An upcoming paper emerging from a collaboration of several European groups provides yet another example of the PDZ domain's allosteric potential bearing fruit, in this case in the regulation of a protease. The article is open-access, so go ahead and open it up in another window to follow along.

Krojer et al. are investigating the heat response in bacteria. Just as your body responds to ambient warmth (by sweating, etc.), bacteria have several systems to help them cope with high temperatures. These include systems for folding proteins that have lost their shape due to the heat, and also systems that degrade proteins when the refolding system can't keep up. A protein that breaks down other proteins is called a protease, and enzymes of this type are widely used in nature (blood clotting, viral maturation, digestion, etc.). Because of their destructive potential proteases are often tightly regulated, as is the case with the protease in this article, DegP. In fact, DegP can also serve as a refolding protein (or chaperone)—some regulatory mechanism causes a switch between these functions. DegP is an E. coli enzyme, but has a similar architecture to some human enzymes linked to diseases that involve protein misfolding and aggregation.

Krojer et al. performed experiments to characterize the proteolytic activity of DegP, mostly summarized in Figure 1. These results indicate that DegP is processive, i.e. that it makes many cuts on a target rather than just one. Using mass spectrometry the researchers determined that the targets got cut up into chunks 8-22 residues long, with the most common lengths being 12 and 17 residues. The protease preferred to cut proteins after a valine, alanine, isoleucine, or threonine: these are very common residues in proteins and interestingly most are β-branched. However, when they made short peptides that matched the apparent cleavage pattern, they did not observe any proteolysis.

From this the authors concluded that binding to the PDZ1 domain of DegP was necessary for the proteins to get cut. They performed a series of experiments with longer peptides that showed that a proper binding site 13-17 residues away from the cleavage site was necessary to get proteolysis. Also, the C-terminal residues preferred by PDZ1 are the same as the ones where the protease cleaves. Modeling an unstructured peptide substrate into the known structure of the DegP complex (Figure 4) indicates that a peptide chain ~16 residues long is needed to reach from the PDZ1 domain of one DegP to its own protease domain, and a chain ~12 residues long is needed to stretch to the protease site of an adjacent DegP molecule.

Well, this suggests a tidy little model. The C-terminus of an unfolded protein binds at the PDZ domain and is cut by the protease about 12 or 16 residues down the line. The cut produces a new C-terminus, which binds at the PDZ domain, and the process repeats. In this way the DegP complex processively degrades unfolded proteins and the bacteria are saved from toxic aggregates. But this is not the whole story. You see, it turns out that DegP is pretty efficient at cutting peptides even if the PDZ substrate and the cleavage site are not attached to each other.

The above model suggests two predictions. First, a peptide that binds to the PDZ1 domain (ALE peptide) but cannot be cleaved by the protease should inhibit the proteolysis of an unfolded protein. Second, the activity of DegP towards a substrate that is too short should not be affected by adding an ALE peptide. However, when the researchers in this case performed these experiments the results were quite different than expected. The ALE peptide slightly activated the degradation of an unfolded protein, and enhanced the cleavage of the short peptide 50-fold. Further experiments indicated that the binding of a peptide to the PDZ1 domain of one DegP molecule activated the protease of that molecule and one neighboring molecule of the complex.

So now the model is more complex, but also much more interesting. Binding of an unfolded protein's C-terminus to the PDZ1 domain does help generate the processivity and molecular ruler effects mentioned previously. But this binding also allosterically increases the intrinsic rate of proteolysis in nearby protease subunits. The effect is a sort of positive feedback mechanism that keeps the protease running until the whole target protein is degraded. This adaptation means that the protease can activate rapidly when there are a lot of free termini floating around, but won't go chopping up every flexible loop it comes across.

The precise mechanism by which binding at the PDZ site activates the protease is beyond the scope of the present study. It may be that the networks suggested by previous research do not come into play in this instance. Looking at the biological complexes predicted from the crystal structure (explore it at the PDB) I would guess that binding-dependent remodeling of the strand linking the protease domain to PDZ1 may be responsible, rather than the dynamic networks. Future experiments will probably address this allosteric mechanism.

1. Krojer, T., Pangerl, K., Kurt, J., Sawa, J., Stingl, C., Mechtler, K., Huber, R., Ehrmann, M., Clausen, T. (2008). Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins. Proceedings of the National Academy of Sciences 105(22), 7702-7707 DOI: 10.1073/pnas.0803392105 OPEN ACCESS

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Diseases and cures in news and advertising

You've certainly seen some of the ads before, selling drugs that cure restless legs syndrome or erectile dysfunction. You've seen the news stories too, with credulous reporters blindly parroting news releases or self-promoting scientists without actually assessing evidence or costs. Companies have been accused of selling diseases at the same time they sell the cures, and the news media has historically done a poor job of educating the public about the true value of new advances in science and medicine. Today in PLoS Medicine, a pair of articles look at corporate and media disease-mongering, and the quality of medical reporting in general.

The first of these articles is an essay by Ray Moynihan and colleagues stating that disease mongering has entered the global health debate. Disease mongering—the creation or promotion of a diagnosis—is something you've probably suspected when watching those commercials, and it's a perfectly understandable strategy. Research into "blockbuster" drugs for major diseases often turns up medicines that fail at their intended task but have some other benefit (Viagra is a famous example of this phenomenon). Companies attempting to recoup the research investment have a substantial incentive to grow the market for these drugs. Even if there is a small group of patients for whom, say, restless leg syndrome is a significant problem, the attempt to expand this market to those for whom it is merely an occasional annoyance is disease mongering. When this tactic succeeds, people end up buying medication they don't really need, which has adverse economic consequences, not to mention the personal harm that may be suffered due to occasionally severe side effects or adverse interactions with other drugs.

The essay follows up a conference and special issue of PLoS Medicine from two years ago on this subject. The authors are pleased to report that several newspaper articles and websites are now taking notice, but this seems too optimistic to me. It is encouraging that we've seen greater awareness of disease mongering in recent years. The authors, however, are unable to point to any significant regulatory progress. A few newspaper articles and a couple of little-known websites are hardly enough to combat a pharmaceutical industry that is dedicated to selling drugs that may have a small natural audience. If we are to have a true national health care coverage program it will be essential to take regulatory steps to prevent the creation or expansion of diagnoses to justify the existence of drugs post facto. In light of the silence of regulatory bodies, even the modest optimism of the essay seems unfounded.

The other article comes from the creators of one of those little-known websites, which I hope will become better known, HealthNewsReview.org. The way the system there works is that three reviewers (one of whom is always this article's author, Gary Schwitzer), evaluate health news stories from television, newspapers, and wire services on the basis of ten criteria ranging from whether the story adequately discusses costs to whether independent sources were used. In this article, Schwitzer reports the results of a survey of 500 stories from US media sources, and the results are not particularly encouraging.

The frequency of particular criteria being met indicates a strong sensationalistic bent in American media. High percentages of articles established the novelty of the approach and discussed its availability. However, less than 40% of the articles did a satisfactory job of discussing the quality of the evidence, the existence of alternative options, or quantifying harms and benefits associated with a new treatment. Less than a quarter adequately discussed the cost of the new treatment. To my mind this speaks to a certain amount of hype. As Schwitzer notes, some of these problems are due to constraints of space and time—in a limited number of paragraphs it is far easier to convey that a novel cure has been discovered than to realistically describe the limitations of the study or the expense of the medicine. Moreover, many of the writers simply lack the expertise to interpret findings, or the resources to find experts to give a second opinion.

This is not entirely the fault of the media, however. Scientific journals, research corporations, and universities contribute by providing sensationalistic press releases that serve as the starting point (if not the entirety) of many of these stories. With commercial interests this practice is standard, though hardly worthy of celebration. In my opinion, however, it is inexcusable for universities and scientific journals to adopt this approach. The reporting of scholarship ought to be about truth, not buzz.

The presentation of results in this article seems somewhat incomplete. I would have especially liked to see a histogram showing the frequency with which a single article fulfilled multiple criteria. Do articles generally fall near the extremes, or do the results more resemble the familiar bell curve? Appropriate policy recommendations may differ significantly depending on this distribution. It would also be valuable to know the track records of particular publications: this data is not available in the article or on the website, yet it could be a valuable tool in putting pressure on editors and managers to give writers the space, resources, and training necessary to produce quality health reporting.

A further weakness of this study is that it does not address cable news or alternative media outlets such as websites and blogs, from which a significant proportion of Americans now get their news. This is especially incongruous in light of the fact that HealthNewsReview.org is itself an internet resource. To be fair, many news websites simply take their stories from the AP feed, which the study examined, and studying all these varied sources would have been an immense task. However, it seems obvious that a comprehensive evaluation of all media was never an achievable goal. Knowing that, it would have made more sense to select the broadest spectrum of types of sources, rather than intensively scanning several very similar sources. It is to be hoped that HealthNewsReview.org will expand its examination into these areas soon.

Sensationalism and sloppiness in scientific reporting represent a failure of journalism because they create a misinformed public. Moreover, overhyping of predictions and results that are often quite equivocal in reality contributes to mistrust of scientists and doctors. Scientists and journalists both must do a better job of communicating the real limits and implications of scientific findings, rather than casting all results in the most positive or revolutionary light. Hopefully HealthNewsReview.org and similar initiatives in other countries will help them do that.

PLoS Medicine is an open access journal, so you can check these articles out for free. Also check out the commentary from the PLoS Medicine editors.

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Called to Arms: Resonance

Steve Gaynor posted a Call to Arms a few days ago, asking his readers to produce:

...a decentralized game design symposium; a call for new takes on interactive expression. If we've succeeded by now in conveying feelings like "exhilaration," "fear," and "victory," and conflicts such as "individual power vs. strength in numbers," "man vs. rule system," "entropy vs. order," and "good vs. evil," the Call to Arms focuses on some more elusive aesthetics.

He explicitly asked people who aren't in the games industry to contribute, so he has only himself to blame for the following proposal, entitled Resonance, for a oratorical simulator.

Resonance takes place throughout the history of a city-state. The particular kind of setting (fantasy/sci-fi/kittens) doesn't particularly matter because the idea is to tap into the universal aspects anyway. On each mission the player become the Muse of a Hero, whose task it is to guide the city through a crisis. The Hero comes with his own point of view, and strengths and weaknesses in communicating to particular demographics. The player's task is to preserve the city, and his only means of doing so are telling the Hero when and where to speak, and providing inspiration for the Hero's speeches.

Each crisis poses a sort of yes/no question: should the nation accept a large influx of refugees, should it go to war against an evil nation that does not threaten the city directly, should workers form labor unions, should income be radically redistributed, etc. By adjusting his strategy the player can allow either of the propositions to "win". The Hero's position is not always "right". Indeed, were I making this game I would have at least one mission where the Hero's choice is quite obviously wrong.

The Hero works his magic through speeches. In a speech, the Hero supplies the main talking points, but on his own the speaker is very dry and dull. Without inspiration from the player, his speeches suck the spirit right out of an audience. The player's task is to provide images that fit the speech and raise the audience's spirit. In doing this the Hero's influence (and the size of his future audience) is increased, listeners convert to his position, and those who are unconvinced become more open to it. At the same time, there will be an Opponent moving around the city trying to convince people of the other side of the argument.

Usually several inspirations will be suitable for each talking point, but they will affect different properties of the audience. I think a 3-4 point matrix like Fear, Anger, Pride, Acceptance would be appropriate. The city is divided into different districts in each era, with different demographics and varying levels of these characteristics. Harsh, divisive rhetoric lowers your side's Acceptance (making them more resistant to conversion) and increases their Anger, making them less likely to attend opposition speeches. However, among the portions of the population that are uncommitted or opposed to the Hero's position, this kind of rhetoric will increase their Fear and decrease their Acceptance, making them more difficult to recruit. On the other hand, inspirations that stress unity would tend to increase Pride of the people on the Hero's side and increase the Acceptance of other groups. However, in an area that already has low Acceptance among the Opposition, a good, harsh speech may provoke Fear that drives them to pay more attention to the Hero.

The strategic task of the player is a balancing act of trying to choose the right kind of speech to give at the right time to the right people, so that the city does not descend into a panic or widespread rioting. When Pride and Acceptance are low, and Fear and Anger are high in any group except those who are undecided, violence can erupt. Usually this will only happen in one district at first, if left alone the violence will spread. Fear and Anger will rise for all groups in neighboring districts. If your side riots, the Pride of your allies in other districts will decrease, while the Acceptance of the Opposition will decrease. So the Hero must calm the violence by giving speeches. If his influence is too low (from making poor speeches earlier), however, nobody will listen to him. The player has to keep the Hero popular, so just flubbing all speeches outright isn't really an option. However, flubbing a few key speeches may work. Having the city descend into riot is the only way to "fail" a mission.

The tactical task of the player (in speeches), is to choose the right inspiration to go with an idea, and also to time the delivery of that image correctly. The spirit of the audience will rise and fall during the speech, rising when the right inspiration is used and falling as the Hero goes over the nuts and bolts. The player has to choose the right time within each talking point to apply the inspiration, and also when to use more powerful vs. less powerful inspirations. So this could be implemented like a rhythm game of some sort. For instance a player could use gestures with a Wii-mote to set up the speaking cadence and then some button combo to use the inspiration at the right moment.

The mission hub takes the form of a giant, labyrinthine library. The character starts in the deepest part of the library and must find his way out. As he moves through the library, the character finds two kinds of books. Opening "Histories" initiates new missions, and when a History is completed a nearby door unlocks, allowing further exploration. Also, the character can find "Literature" books that give him new inspirations. Because the "combat" is oratory, these will obviously include important speeches ("I Have a Dream", the sermon on the mount, "Never, never, never" etc.). However, since the player's task is to supply imagery and inspiration they should also include poems (Dwarf Birches, If) and excerpts from plays (Henry V) and novels.

Different outcomes to the missions unlock different doors and thus different routes through the library. The inspirational Literature available to find along the way (and the powers to be used in combat) therefore depend on the outcome of the missions. In addition, the Hero of each mission becomes available as an inspiration power for future missions; the strength of this inspiration will depend on the mission outcome. This need not be linearly related to whether he convinced people of his position. Losing to the opposition for strategic reasons while performing well in individual speeches may result in a powerful inspiration; succeeding strategically while barely scraping by in speeches may result in a weak one.

Unlocking the final door of the library releases the character as a citizen into the city he has created through previous missions. The character of the city will be decided by the character as well as the facts of the decisions made. So, if the city accepted the refugees, then traces of that race of refugees will still be physically present. In addition, the inhabitants of the city will tend to be kind and helpful. If you opted to take on the evil city, they will be proud of their city but have a militaristic streak. The city will be facing one more great moral crisis, but one that is a trick question, in which one of the alternatives seems quite reasonable and comforting but is, when properly considered, a path to destruction. The player must now serve as his own muse, working up influence from street level so that he can convince the city to do the right thing. This will be easier or harder depending on the ways that the previous missions were performed.

Having only one Hero position per mission would simplify the programming task while confronting the player with the fact that people can be just as honestly passionate about a proposition he disagrees with. If I were to pick one feeling out of Steve's laundry list that I meant the design to speak to, it would be vindication (in seeing the city you created through your actions as a muse). The individual missions could be tailored so that they speak to several of the conflicts he proposes, though the conflicts of Pragmatism vs. Romanticism and Tradition vs. Progress are clearly built into the fabric of the design (such as it is).

Well, this may be the worst idea for a game ever, but I had fun thinking about it, so thanks, Steve. Readers should check out the post I linked to above for some of the other entries. All of them are interesting and would make good, or at least intriguing, games.

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[Insert poop joke here]

It's rare for me to be reading just one book at a time. Usually I bounce back and forth between at least two—one that I read at my desk or on the couch, and one that I read in my "inner sanctum". It's an unusual book that has me so interested that I'm willing to go searching for it every time I want something to read (or need to use the can). Yet for the time it took to read it, Carl Zimmer's Microcosm: E. Coli and the New Science of Life was essentially glued to my hand. Perhaps it would have been more appropriate, given the subject matter, to only read it on the throne, but it was just too good to be read in mere 15-minute snippets.

First isolated from baby diapers by a German physician in the 19th century, Escherichia coli (and its Mr. Hyde, Shigella) is most famous today for making people sick. Inside our bodies E. coli is a pioneer—one of the first species of bacterium to set up shop inside us, though hardly the last, and in normal circumstances it can provide a protective effect. At the heart of this seeming contradiction is the fact that E. coli has enormous diversity. Much of the fun in reading Microcosm lies in coming to understand how that could be so, given that this lowly microbe mostly reproduces by binary fission.

As a structural biologist, I frequently use liters of E. coli—primarily a strain called BL21(DE3). One thing Zimmer notes is that E. coli is absolutely essential to modern molecular bioscience. Its ability to produce the DNA and proteins we need for research makes this lowly bacterium one of our greatest investigative tools. Biomolecular NMR as we know it owes as much or more to the humble microbe (and its willingness to grow thickly in nutrient-poor media) as it does to men like Wüthrich and Bax.

Those who, like myself, primarily know E. coli as a tool for bioscience will recognize several names along this little tour, and learn a great deal about how it came to be such a powerful resource for us. Many will be surprised to learn how controversial some activities we take for granted (transfection of exogenous genetic material into bacteria, for example) were 20-30 years ago. Readers who have very little biology background need not fret, of course. Carl Zimmer is an excellent writer who introduces all the material carefully. Only some very basic knowledge about DNA, proteins, and bacteria is necessary. Zimmer will carry you the rest of the way, from bacteriophages and flagella all the way to synthetic biology, GM crops, and gene circuits.

Whether you work with E. coli every day or just cringe when you hear those words on the evening news, Microcosm will be an engaging, rewarding, and informative read.

While you wait for a chance to go to the bookstore (or for delivery, if like me you use Amazon all the time) check out Carl Zimmer's blog, The Loom, at ScienceBlogs.

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Gold dust vs. the virus

A recent episode of South Park featured a story in which two of the main characters got infected with HIV and discovered that the cure for AIDS is an injection of about $200,000. As any viewer of the series would expect, the episode is crude and vulgar, and it wobbles to and fro over the line between humor and offensiveness. Yet the episode might also turn out to be oddly prescient, if research described in an upcoming JACS article bears further fruit. As it turns out, researchers from UNC, the University of Colorado, and NC State have had some success in inhibiting HIV activity using drug-coated nanoparticles, made out of gold.

The approach Bowman et al. use is based on the idea of multivalence, which is the operating principle of Velcro. A single hook-loop interaction between two pieces of fabric usually isn't enough to keep them fastened together. However, by having a large number of relatively weak interactions a strong connection can be made. Many biological systems make use of the same principle, using many weak interactions between repeating units to produce high overall affinity. The researchers set out to apply this idea to medicine, using many copies of a low-affinity drug attached to a nanoparticle.

The drug the authors used is based on a compound designated TAK-779, which is effective at preventing HIV virions from fusing with T cells, but also has some unpleasant properties for patients. The authors lopped off the part of the molecule that causes these problems, but doing so also removes most of its ability to fight HIV. So, they linked this new compound (called SDC-1721) to the gold particles at a ratio of about 12 molecules SDC-1721 per particle. In cultured cells, the nanoparticle-linked drug had an IC50 similar to TAK-779, even though SDC-1721 by itself was totally ineffective at preventing infection. Cutting the number of SDC-1721 molecules per particle to ~1 removed the inhibitory effect, proving that the multivalent approach was critical.

This is of course no demonstration of in vivo effectiveness, and there's no telling whether the nanoparticle will have side-effects that are better or worse than TAK-779. However, if this initial success is borne out by further trials this may be a promising angle on treatments. One of the advantages to this approach is that it has some ability to counter resistance built into it because of the multivalent binding. Even if a virus evolves a lower affinity for the drug, the weak binding of many ligands, and the increased effective local concentration of those ligands, may be enough to rescue inhibitory activity. Injecting yourself with money is no way to cure anything, but it is possible that in the future we will attack viruses by injecting patients with (drug-laden) gold.

1. Bowman, M., Ballard, T.E., Ackerson, C.J., Feldheim, D.L., Margolis, D.M., Melander, C. (2008). Inhibition of HIV Fusion with Multivalent Gold Nanoparticles. Journal of the American Chemical Society DOI: 10.1021/ja710321g

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You're like a tiny god!

By now many of the nerds in my audience have heard about Foldit—the server seems to be wanged, so clicking on that link may not do anything for you. The idea behind Foldit is to use the processing power of human beings to address the protein folding problem in the context of a game. Despite many years of trying, predicting the fold of a protein and moving a random coil into this shape is a task that computers typically find very difficult. Distributed computing efforts, like Vijay Pande's folding@home or Rosetta@home, are one way of addressing this problem for some limited cases. Foldit, from David Baker's group, is conceived as a way to train the ROSETTA program to get better at moving structures to their ultimately predicted shape. This may sound complicated, and the system isn't perfect, but almost anyone can play and it's surprisingly fun.

The game is relatively simple to use, if a little flaky. You are handed a peptide chain with secondary structural features already formed. You can move these motifs into the form of a packed structure by manually moving them around, by letting ROSETTA wiggle them freely, or by giving ROSETTA general directives about where to move the elements during this wiggling. Directing the automated movements is pretty seamless, though clashes sometimes have a way of causing the whole protein to blow up. Many of these clashes can be resolved by "shaking" the side chains, also a relatively seamless automation.

However, your manual control is limited and occasionally awkward. Sometimes, Foldit just won't move a side chain where you want it to go, and at other times it will persistently rotate the wrong angle. As I noted above, the secondary structural elements are pre-set, which is okay for helices but seems a little off in the case of strands. Also, many of the loops are much more rigid than expected. Many of these problems could be solved by context-sensitive menus that allowed you to select and rotate a particular bond angle; this could also help with the axial rotation of β-strands, something I very much wanted to do in two of the puzzles. I have yet to see hydrogen bonds or charge-charge interactions between side chains, and indeed the game has comparatively little information about side chains generally. In default mode, unless they clash, they're visible only as little nubs with a faint glow to tell you whether they're hydrophobic or not. A lot of this may be simplification on the part of Foldit to prevent "impossible" conformations, but this is a limitation the creators should explicitly try to avoid. Avoiding what it feels to be impossible may be one of ROSETTA's flaws, after all.

Playing Foldit is an interesting experience, and the puzzles are fun and challenging without requiring any detailed knowledge about biochemistry. People who do know something about biochemistry, however, may find themselves frustrated by their inability to make moves that seem sensible. And, the simplicity means that people who don't know any biochemistry probably won't learn anything lasting from Foldit. The controls are a little wonky and at times seemingly unresponsive. Still, this is just the open beta version, so hopefully some of these minor annoyances will be fixed going forward. Once the furor dies down (in about a week or so), you should head over to the site and see if you're interested. The game is free to download, but requires an internet connection.

In related news, an article by Baker that I covered earlier concerning the use of ROSETTA in combination with directed evolution to produce relatively active enzymes has been published in this week's edition of Nature. It's an interesting article, and definitely worth reading if you have an interest in protein engineering.

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It's just lines and colors

Anyone picking up No More Heroes will wonder just what the hell it is that he's playing, because like creator Suda Goichi's previous game killer7 it defies easy categorization. That prompts a question, though: why should we want to categorize these games? Does figuring out what bin to drop a game into make the world easier to interpret, or do our categories, and the conventions that come with them, impose meaningless constraints on our appreciation and enjoyment of art? Early and late in No More Heroes Suda breaks the fourth wall—a sign that the game is trying to say something about its own medium. Suda seems to be illustrating the limits of video games—the borders of what they can do, and the borders of what we allow them to do.

Spoilers follow; abandon all surprise, ye who enter here.

The protagonist of No More Heroes, Travis Touchdown, is a scrawny, unattractive pervert with a bad haircut and questionable hygeine who apparently gets plenty of fiber in his diet. Travis is a confirmed otaku, and his motel apartment is filled with collectibles from an anime called "Pure White Lover Bizarre Jelly" that is clearly a fusion of moe shoujo (behold the cute magical girls) and mecha (they pilot enormous robots called 'Glastonburies') styles. In addition, he's obsessed with lucha libre wrestling. Having won a "beam katana" in an online auction—and if you can't guess what that's supposed to be you need to stop reading—he is entered in a competition to become the world's #1 assassin by killing everyone ranked above him, a task he engages with gusto, for reasons that are somewhat mysterious even to him. Basically Travis is a dork who has been handed a real functioning lightsaber and told to go kill.

And kill he does. About half of the game involves taking on hordes of enemies using the beam katana and wrestling moves. Depending on the context one or the other will be more effective, but the ultimate killing blow always comes from the katana, ending your enemies' lives in a shower of blood and cash that one reviewer compared to a gory slot machine. At the end of certain missions Travis finds a 'ranking battle' against one of the better assassins. The grunts along the way have little personality, but each of these ranked assassins has something interesting to set him or her apart.

I would love to say that all this constitutes some kind of commentary on violence in video games, but I'm not even sure that's possible anymore. A kind of Poe's Law has come into effect, in that it's no longer possible to distinguish a game that parodies excessive violence from a game that celebrates it. Video game violence has progressed beyond the possibility of irony or satire, and maybe the fact that No More Heroes reinforces this idea is kind of a message in and of itself.

When confronted with the idea of a person being cut in two vertically and exploding in a shower of blood and coins one is tempted to say that boundaries are being pushed, but it's important to understand the degree to which the combat emphasizes constraint. I don't mean a physical constraint here, but rather an intellectual one. Travis' primary attacks and defense (his 'Force') are all derivative of existing entertainments (Star Wars and wrestling), and his special attacks are mostly named for the characters in the aforementioned "Bizarre Jelly". The Assassin's Association is his way out of his fanboy lifestyle but at the same time his very mind is locked into shape by the mass-market pap he has consumed. What keeps him trapped in his lifestyle is not the absence of fame or money: it is his inability to imagine anything else.

But of course, not all of the game is killing. In order to reach the ranking battles against more qualified assassins Travis needs to gather quite a lot of money, and to this end he works a variety of demeaning part-time jobs and simple assassination missions. To get to all of this work and shopping he tools around the game's town, Santa Destroy, on his monster motorcycle.

The mission-based structure and ability to roam freely around town certainly recall Grand Theft Auto, but the resemblance here is only skin-deep. The town is plenty large, but spots for actual activity are rather few and far between. Indeed, about half the town has essentially nothing to do at all beyond some dumpster diving. As for the people walking around, you can't speak to or attack most of them. What you can do to them—run over them with the bike—seemingly neither harms them nor draws the attention of law enforcement to you. In fact, though you'll encounter plenty of police cars, nobody seems to care that you're running over pedestrians and lampposts, or leaving a trail of bodies that have been bisected in interesting ways.

What we're confronted with here is the structure of a Grand Theft Auto without any of the texture. If you've ever seen one of Mondrian's compositions this feeling may be familiar to you. Suda has tossed in the lines and the colors, but none of those things are actually being built into the representation you expect. A Mondrian has content, and so does No More Heroes, but the sparsity of identifiable content has the effect of emphasizing the structural aspects of whatever it is that you are dealing with—in this case, a game.

Games are fundamentally constrained systems. Consider Grand Theft Auto itself for a minute. It's widely and rightly praised for its flexibility and content, but there's no escaping the fact that it's a game about a violent criminal doing things that are violent and criminal. Niko Bellic can't hook up with a handsome French man named Pierre, move to Provence, and start over as a vintner. No, Niko's a criminal, and that's the whole idea of a GTA game. The freedom of the sandbox games is an illusion, and No More Heroes loses the inner texture that creates that illusion, leaving behind the framework to remind us of what we expected to see. Suda seems to be arguing that realism in games isn't necessarily a valid pursuit. Their nature precludes them from matching reality, and even if they did match it, they would cease to be art and become something else.

"The expression of reality cannot be the same as reality."—Piet Mondrian

The second idea, that our expectations constrain our own approach to media pops to the fore in the game's finale. There is another nod to the creative prison, in the form of a mysterious masked man stating that he is Travis' father. There is also a reference to the external limitations imposed by censors and corporate development cycles. But the principal source of constraint that Suda points out is us, the conventions that are enforced by our expectations.

This underlies all the enormously convoluted twists in the storyline and the amnesia that we only learn Travis has as soon as he overcomes it. As Travis' family multiplies rapidly to include an unsuspected brother and sister (both of whom he must kill), the whole story of the game is thrown into doubt, and seemingly discarded characters inexplicably reappear. None of it really makes very much sense, and that's kind of the idea. The plot developments have been thrown in to satisfy convention, without the narrative fabric that would allow one to make sense of them. The point here is to emphasize that features which rightly ought to be part of the content have been forced to become part of the structure by our expectations.

Video games are constrained by their programming, but also by our expectations and the conventions those expectations enforce. In giving us the lo-fi accessory graphics and textureless structure of Santa Destroy, Suda embraces the former; the gonzo finale seems to be his way of lampooning the latter. Taken as an expression of any particular genre, No More Heroes certainly fails to impress. But in a way it seems this is almost the point. The genres and their conventions are stale, and no level of graphical power or snazzy controls can disguise that fact. Nor can we get around the creative problem by smashing genres together.

In many ways, the array of games on the market resembles Travis' collection of Luchador masks. Each of them has different colors and additions, but in the end there's no escaping that they're all essentially iterations of the same thing. That's not just because designers and the corporate suits who write their paychecks are lacking in creativity. It's also because the consumer rarely tolerates or accepts something that doesn't fit preconceived notions of genre. In the end, No More Heroes is a game about games, an acknowledgment of the limits of what they can do, and a protest against the limits of what we let them do.

There are a number of other takes on No More Heroes that I quite liked.
Yahtzee's review is hilarious but NSFW.
Steve Gaynor has a comprehensive review.
The Brainygamer compares No More Heroes to the French New Wave and also serves up a more conventional homage.
Insult Swordfighting has a good take, too.
In theory, if you are reading this review, you already subscribe to those blogs. If not, then why aren't you subscribed to those blogs?

Comments on my view or recommendations of additional alternative interpretations are certainly welcome.

More good reading on No More Heroes
Dan Bruno at Cruise Elroy talks sex and violence. He also has a postmortem with extra linkiness.
Schlaghund has a view very close to my own.
Cowzilla has an article at Destructoid that also asks what No More Heroes is saying about us.

Read the rest...