Proteins don’t get a lot of publicity, at least not compared with their glamour-puss cousins DNA and RNA. DNA, ensconced deep in the nucleus of cells, is the fountainhead, the living library of genes that embodies the very blueprints of life. And DNA begets RNA, the intrepid genetic messenger, braving the wilds of the cell to deliver DNA’s instructions to outlying factories that translate the blueprints into building materials-- that is, into proteins. Proteins just do all the work: they assemble, modify, and maintain the cells. True, without the efforts of these blue- collar laborers there would be no life at all, but proteins seem to lack the flash that has made heroes of their genetic kin.
But at last proteins are coming into their own. Proteins are amazing and subtle, says Peter S. Kim of the Whitehead Institute for Biomedical Research and the Howard Hughes Medical Institute at MIT. They’re finely tuned machines. I try to understand how they do the marvelous things they do.
Kim is not alone. His field of protein biology is flourishing as never before. Kim himself--at 37, he’s one of the field’s hottest young stars--has not only made discoveries that illuminate the fundamental nature of proteins and their relationship to diseases but has also identified an intriguing protein mechanism that may explain how viruses like flu and HIV manage to work their way into our cells. In doing so, Kim has perhaps provided a key for stopping those viruses in their tracks. The secret lies in the shapes proteins take when they fold.
Protein folding is one of the marvels of nature. When proteins roll off the cell’s assembly lines, they are nothing more than long chains of amino acids. Amino acids come in 20 varieties, and proteins typically contain between 100 and 10,000 amino acids. The acids function as a kind of alphabet, spelling out the form and function of the protein. Just as the 26 letters in the English alphabet can be arranged to spell a mind-boggling collection of words, the 20 amino acids combine to form tens of millions of proteins across the range of organisms on Earth. The human body alone contains some 50,000 kinds; among the structures they’re responsible for are muscle, skin, hair, cartilage, antibodies, enzymes, and hormones, to name just a few.
But as newly minted one-dimensional chains, proteins are useless. For them to assume their myriad forms and carry out their vital duties, they must bend and twist into intricate three-dimensional shapes held in place by chemical bonds. Imagine crushing a length of yarn in your hand. The tangled mass resembles a folded protein. Some proteins coil into loops or spirals, others bend into hairpins or press into pleated sheets resembling accordions; any given protein may contain several of these shapes, in unique and specific arrangements. Shape promotes function. For example, the nooks and crannies in the folds of a digestive enzyme trap starch molecules, which can then be placed near chemicals that break them down into sugar. Similarly, bacteria and viruses fit snugly into the folds of antibodies, which hold them tight while summoning help from other immune system defenders.
For the past seven years, Kim has been focusing his research on one of these shapes, called the coiled coil. Imagine two proteins side by side, both folded into spirals like coiled telephone cords. If the spirals corkscrew around each other, forming a tough cable of coils, that’s a coiled coil. Stable and strong, coiled coils typically show up in structural proteins--muscle, skin, hair, and various filaments--as well as on the surfaces of certain viruses. Nobel laureates Linus Pauling and Francis Crick independently predicted the existence of coiled coils back in 1953. (Later in ’53, Kim points out, Crick and James Watson predicted the coiled structure of DNA, which somewhat overshadowed this one. )
In 1988, Kim started to look for coiled coils in what seemed an unlikely place: a protein that switches genes on and off to regulate when other proteins are produced. Until then, coiled coils had been seen primarily in structural proteins, he says. We thought it would be interesting if this regulatory protein turned out to contain a coiled coil.
The protein carries out its gene-regulating task through cooperation--two proteins link together to act as one functioning whole. Combining forces, the partners literally squeeze DNA between them like a wrestler applying a scissors hold. When Kim began his detective work, it was thought that these two proteins were spirals connected by amino acids called leucines. The leucines supposedly formed a rough ridge of knobs along one side of each spiral. As the two proteins abutted, the leucines interlocked, the knobs on one spiral wedging between the knobs on the other like teeth in a zipper. In effect, then, the two proteins zipped firmly together--thus the term leucine zipper, coined by their discoverer, Steven McKnight of the Carnegie Institution in Baltimore.
McKnight didn’t actually see these zippers, of course. Proteins are too small to see, even with an electron microscope, so researchers must rely on a variety of indirect methods. The most definitive, and difficult, involves bouncing X-rays off a crystallized version of the protein and then, with the aid of computers, constructing its shape from the pattern made by the deflected rays. But McKnight predicted the leucine zippers and the spiral structure of the proteins based on the sequence of the amino acids that constituted them. It’s brilliant work, says Kim. It has very well thought out predictions and logical deductions. Except that they concluded the structure was not a coiled coil. Kim and his graduate student Erin O’Shea suspected otherwise.
To examine the proteins, Kim added an innovative touch: he made a model. In the laboratory, he and O’Shea constructed fragments of the regulatory proteins by linking amino acids in the same order as that of the real thing. These protein pieces, or peptides, were much easier to handle than the entire thing and allowed Kim to focus on the precise section of the protein he suspected was a coiled coil. When the researchers plunked their homemade peptides into a saline solution resembling the soup inside a cell, the amino-acid minichains coiled into spirals and twisted around each other like two snakes intertwining--in other words, they formed coiled coils. But they didn’t exactly zip together. Rather than interlocking, the leucine knobs on the side of one coil snapped into knobs on the other coil like a bunch of children’s toys.
In 1989, Kim published his results. Two years later, with protein crystallographer Tom Alber, who’s now at the University of California at Berkeley, he obtained a high-resolution image of the structure by X-ray crystallography, the equivalent of making a detailed three-dimensional map. It was the first such map of an isolated coiled coil. Kim and his colleagues had found something previously unknown in nature: a regulatory protein coiled coil held together by knobs snapping together. People have asked me why we didn’t change the name, says Kim. Well, we thought McKnight should get the credit for discovering the characteristic shape of the protein. And compared with ‘leucine zipper,’ a new name wouldn’t have stuck anyway.
The discovery became all the more pertinent a year later when Kim and O’Shea turned their attention to a regulatory coiled coil made up of two proteins called Fos and Jun. In this case the protein partnership facilitated a deadly operation. Fos and Jun are oncoproteins--proteins produced by cancer-causing oncogenes. They work together to disrupt the normal expression of DNA and provoke cancer. Kim and O’Shea built peptide models of the proteins and found that this sinister coiled coil, too, was joined by the same kind of snapping linkage.
Understanding how these proteins come together was a central question for us, because it clearly had important implications for health, says Kim. Now we know that to disrupt the interaction between Fos and Jun, one would want to prevent the linkage. The region where the oncoproteins join could therefore be an important target for anticancer drugs. Kim’s peptide models themselves might provide a means of attacking them.
If you were to add a lot of these peptides to a cell, you would expect them to interfere with the coiled coil, he says. Sometimes the synthetic Fos peptide would hook up with the Jun protein, and the synthetic Jun peptide with the Fos protein, preventing the two proteins from seeking out their natural partners. And if enough Fos and Jun look-alikes invaded the cellular ballroom, the real proteins would rarely find each other for their deadly waltz.
Kim’s Fos and Jun findings, published in 1992, prefigured an even more dramatic discovery. Once again the impetus came from coiled coils. Kim had been thinking about the general question of how to predict a protein’s 3-D shape by knowing the order of its constituent amino acids. I should be able to give you an amino acid sequence, and you should be able to tell me what the structure will be, Kim says. One place where we think we can do that is with coiled coils.
Coiled coils lend themselves to such prognostication because they are made up of relatively simple repeating sequences of amino acids. In fact, in 1991 biologists at Princeton had written a computer program that they claimed could plow through the amino acid sequences of an enormous number of proteins and pick out the ones that were likely to be coiled coils. Kim and graduate student Chavela Carr decided to test the program on proteins whose 3-D structure had already been determined by X-ray crystallography. We took the sequences the program predicted would be coiled coils, compared them with their crystallographic structure, and checked to see whether they really were coiled coils, says Kim. Unfortunately, all too often they were not.
However, one of the structures that was correctly predicted occurred on the surface of a flu virus, in a prominent feature known as the hemagglutinin spike. If it were possible to explore the surface of a flu virus in a submicroscopic spaceship, you’d behold a fantastic landscape. The surface of the virus is punctuated all around with protein spikes and looks remarkably like the business end of a medieval mace. The most common of these spikes is hemagglutinin (HA for short), which was mapped in 1981 by Harvard crystallographer Don Wiley. If you flew closer, you’d see that the HA spike consists of three spiral peptides that corkscrew around one another to form a coiled coil. At the top of each coiled peptide sprouts an unfolded amino acid chain that loops down the side of the coil like a drooping vine. At the end of each of these three protein vines is another spiral segment, shorter this time, and then finally a short chain that horseshoes back up like a hook. Surmounting the entire structure, like puffy clouds circling a mountain peak, are three protein balls.
Researchers knew that when a flu virus approaches a target cell-- in the nasal passage, throat, windpipe, or lungs--the cell, aware that the virus is lurking nearby, swallows it into a pocket called an endosome. The cell tries to destroy the invader by breaking down and recycling its proteins and other building blocks. But instead of being digested, the virus strikes. Its peptide hooks sink deep into the wall of the endosome, allowing the virus to fuse with the cell, deposit its genes inside, and begin the process of infecting its devourer.
What wasn’t known, however, was how those hooks, which hung at the end of the limp vines, got anywhere near the endosome membrane far away. And as Kim and Carr compared the amino acid sequence from the computer program with the map of the real thing, that ongoing mystery was soon joined by a new one: the sequence didn’t correspond to the section of the HA spike that the crystallographic maps had shown to be a coiled coil. Rather, it was identical to the amino acid sequence of the drooping vine. Kim explains: We took the sequence that said coiled coil, went to the hemagglutinin, and said, ‘Wait a minute! That’s not a coiled coil, that’s the loop!’
Was the prediction simply wrong, or was the virus harboring a secret? To find out, Kim again began constructing models, linking amino acids to build copies of the looping vine. Then he dumped the synthetic loops into test tubes containing solutions that approximated the contents of the endosome. Suddenly the hanging loops curled into a coiled coil. The prediction was right after all.
Why, then, didn’t the coil appear on the crystallographic map? Because inside the cell, the loops form a coiled coil only at the moment when the virus fuses with the cell--the moment when the hanging hooks sink into the membrane wall. And within the endosome that moment occurs in a high-acid environment: as the cell tries to break the virus down into digestible bits, the fluid within the endosome becomes more and more acidic. Wiley’s map described an earlier stage, before the virus fuses, and thus a different environment. No wonder the coiled coil didn’t show up.
With that, Kim had the solution to the mystery of how the hooks made contact with the cell membrane. The puzzle was, How does the hook get inserted into the target membrane all the way up at the top of the hemagglutinin? Well, when these loops form a coiled coil, they spring up and drag the hook with them.
Subsequent research in Kim’s lab fleshed out a bizarre scenario. Rather than hanging limply, the loops are actually bent back under tension like springs. What holds them in place? The three protein balls at the top of the HA molecule. When the acid level within the endosome becomes high enough, the balls fall away, releasing the loops. Like sprung mousetraps, they vault up, twist into a coiled coil, and whip the trailing hooks into the cell’s membrane like a harpoon.
Last september, Wiley and his team announced that they had crystallized the HA protein in an environment that had the same acidity as the endosome during fusion. Their new map shows a sprung coiled coil where the loops had been. In October, borrowing a page from Kim’s book of techniques, biologists at Lawrence Berkeley Laboratory and the University of California at Berkeley made peptide models suggesting that once the sprung coiled coil sinks its hooks into the cell membrane, it splays apart, pulling the viral surface closer.
This was, to say the least, a surprising protein. Usually what we deal with are very subtle differences, says Kim. So to have a protein that undergoes this enormous conformational change, literally swinging way, way out--it’s not the kind of thing you bump into every day.
Another thing a biologist doesn’t bump into every day is a way to interfere in a widespread infection process. But knowing the flu virus’s mechanism makes that a possibility. Kim’s strategy of employing synthetic peptides to disrupt the Fos-Jun coiled coil may be applicable here as well- -perhaps drugs that act like these peptides might interfere with the formation of the HA coiled coil. Moreover, it looks as though flu is not the only virus that employs this flamboyant fusion technique. Respiratory syncytial virus, the leading cause of bronchial and lower respiratory tract infections in infants worldwide, may be another. HIV, the AIDS virus, may be one, too. Both viruses contain coiled coils. Kim is now making peptide models of the HIV coiled coil to see whether it too is a loaded spring. Virologists at Duke University have found that, in the test tube at least, peptide models of the HIV coiled coil can indeed stop the virus from infecting. The biotech firm Trimeris, based in Research Triangle Park, North Carolina, is developing the approach for testing in people.
It’s all a testimony to basic research, Kim says. If you had given us a bunch of money to study HIV, we never would have come up with this approach. It’s only because we’re interested in protein folding that we’ve gotten to this point.