A lesson from Botox

There's not much in common between what the Real Doctor studies--ophthalmology--and my chosen field of microbial genetics and physiology. One of these rare commonalities is an interest in Botox. For the Real Doctor, it's a handy tool to paralyze an ocular muscle and cure a case of walleye. For me, "Botox" is botulinum toxin, a poison secreted by Clostridium botulinum, and a nice example of how bacterial toxins work.

Right off the bat, I gotta say that bacterial toxins are amazing. It always gives me pause when I see examples of evolution reaching across domains of life. I mean, it is not too surprising when a bacterium evolves a chemical signal to communicate with another bacterium--after all, they share the same biochemistry. But with toxins, bacteria have evolved a very complicated set of molecules that target very specific proteins on the surface of very specific nerve cells in only a few types of organisms; the bacterium is reaching across domains to communicate biochemically with an alien biochemistry.

That said, there are other reasons to be interested in bacterial toxins. They’re amazingly potent—30 nanograms of botox, or a volume of about one billionth of a sugar cube, is enough to kill a human. Their power and specificity makes them valuable tools for understanding the biochemical processes of our own cells, and some (such as botox) have medical uses. Bacteria, in their diversity, have evolved a huge number of bacterial toxins specific to different tissues in different hosts, but most of these toxins are variations on a couple of themes.

One major class of bacterial toxins can be thought of like nuclear missiles; they require two sophisticated components to do their deadly job. By itself, an A-bomb is not so useful a weapon--unless you deliver it to its target, you will only damage yourself. By itself, a guided missile won't do too much damage to its target--it's merely a bus to deliver the payload. But put together the A-bomb and the bus, and you have yourself a tremendously destructive weapon. The bacterial "A-B" toxins are the same way. The "A" component of these toxins is like the A-bomb--a tremendously destructive protein molecule, but it needs to be delivered to its target. The "B" component is like the missile (or bus), a protein that is by itself harmless, but protects the "A" component and delivers it to its target.

Botulinum toxin is an example of an A-B toxin, with a wicked but delicate A component and a very interesting B component. The bacteria release this toxin in our guts, an extremely acidic environment, and it must travel into our blood and then find a specific molecule on a specific type of nerve cell to do its dirty work. Some recent work by a group at the Sanford-Burnham Medical Research Institute in La Jolla, California, has given us a peek into how the B component delivers the destructive A component to its target, and also--like any good study--raised some more interesting questions.

The researchers focused on the first stage of this deadly missile's trajectory--the trip from the acidic digestive system into the more neutral bloodstream, a hazardous journey that almost no proteins can survive. As long as people have known that botulinum toxin is made of protein, it has been a mystery how it avoided destruction. So, what about B allowed this? By making various modified versions of the B component, they found that only one of the four parts of this carrier was necessary for protectin the A component from acid. Then, since the function of any protein is intimately tied to its structure, they tried to find out the structure of this minimal B and how it fit together with the A. They found a couple of interesting surprises.

First, the B protein is sensitive to acid, just as susceptible to acid as the delicate A component of the toxin. Both B and A, individually, are attacked by acid at a few specific sensitive places, and break into a few distinctive pieces. However, if you put them together in an acidic environment, they laugh it off. The reason is in how these proteins fit together: all the bits that are sensitive to acid are covered up by the way the proteins nestle up against each other. They snuggle against each other to protect each other from harm, a romantic image if we weren’t dealing with deadly poisons.

A second interesting feature came to light when the researchers looked at exactly how these proteins fit together. The researchers were able to recognize the specific parts each protein that touched each other. These parts were notable because of how they responded to acidity: in the harsh of environment of the stomach, these parts of the proteins remained neutral, and helped to hold the A and B complex together. However, when the protein complex was moved to a neutral environment, these parts of the protein turned acidic--enough that they would cut the bonds between the A and the B proteins. The connections between the A and B proteins acted like the explosive bolts that hold together the stages of a missile, holding them together through the boost phase but dramatically separating the two when the booster is done. Indeed, the researchers conclude that this is how the first part of botulinum toxin's journey goes: the minimal part of the B component protects the A component in the stomach and carries it into the bloodstream. Once the two are in the bloodstream, the neutral environment causes B to cut its links to A, and the A component can go on its merry way.

This is all pretty cool. It solves a mystery about how botulinum toxin works, and actually suggest as way that biotechnologists could design protein-based drugs that could be taken orally. If a protein drug could be designed to fit with a carrier similar to botulinum toxin B component, then it could survive the trip through the stomach and be absorbed into the bloodstream, where it could do its job.

However, for my money, the most surprising (and instructive) thing revealed by the structure of the minimal botulinum toxin is that the "A" and "B" components have almost identical structures. Proteins are linear strings of hundreds of amino acids, and these strings clump and fold together in unique and characteristic ways. The structures are often quite complex, with enough helixes and turns and twists that a single protein's structure would resemble a plate of spaghetti, the the entire platefull were one loooong noodle. It is this unique molecular shape that gives each different type of protein the ability to do its specific job--say, disable a nerve cell for the A component of botulinum toxin, or protect another protein from acid for the B component. It is surprising that two proteins that you might expect to have structures as different as an A-bomb and a booster rocket to look almost the same.

Check out this picture: the A component is an orange stringy ribbon, and the B component is a green stringy ribbon. The two structures are superimposed upon each other, and you can see that they are almost identical. (This is just the first third of each of the proteins, but the remaining 2/3 are available, in stereo, at http://www.sciencemag.org/content/335/6071/977/suppl/DC1)

One of the great big hairy problems of modern biology is how proteins--linear molecules--fold up into unique three-dimensional shapes, and botulinum toxin gives us another bracing surprise here. I teach my intro bio students that the "primary structure," or linear arrangement of amino acids in the protein, determines the three-dimensional structure of the protein. A change in the amino acid sequence should result in a change in the three-dimensional structure. I'd expect the botulinum toxin A and B components to have very similar amino acid sequences, given their structural similarity. However, their amino acid sequences are only 20% identical. That's crazy.

The authors, being most interested in biochemistry, don't pursue a Big Question raised by this--how does this evolve? The genes for botulinum toxin A and B components are probably also about 20% identical (mea culpa--I don't have my computer with me, so I have a hard time looking this up. It is left as an exercise for the interested reader). It's possible that two entirely different genes evolved to have such similar structures, but I don't think it's at all likely. More likely, it is a case of an ancestral gene being duplicated, and then each of the two copies undergoing evolutionary change (this hypothesis is weakly supported by the observation that the A and B genes tend to be clustered). These changes altered the sequences of the proteins, while preserving their three-dimensional structures. This would be like sitting down with a sonnet and a thesaurus, and changing 8 of every 10 words. If you were careful, you could preserve the structure, meter, and rhyme scheme of the sonnet, while completely changing the meaning of the poem—and a textual analysis would show no relationship between the source and the end product.

If I could tell these researchers what to do, I would have them gather more structure and sequence information from a variety of related toxin proteins. It would be fun to make a family tree, and see just how much structure could be conserved as amino acid sequence changes.

Shenyan Gu, Sophie Rumpel, Jie Zhou, Jasmin Strotmeier, Hans Bigalke, Kay Perry, Charles B. Shoemaker, Andreas Rummel, Rongsheng Jin (2012). Botulinum Neurotoxin Is Shielded by NTNHA in an Interlocked Complex. Science 335: 977-981.

Peck, MW (2009). Biology and genomic analysis of Clostridium botulinum. Advances in Microbial Physiology 55: 183-265.