Combine three Nobel laureates with one of the world's brightest x-ray sources, and you're bound to get something big — or, in this case, exceedingly small. Using Berkeley Lab's Advanced Light Source (ALS), a highly decorated team of University of Texas Southwestern Medical Center and Howard Hughes Medical Institute researchers determined the three-dimensional structure of a protein that controls cholesterol level in the bloodstream.

Knowing the structure of the protein, a cellular receptor that ensnares cholesterol-laden low-density lipoprotein (LDL), will help researchers understand how LDL receptor mutations promote dangerously high cholesterol levels in some people. And this knowledge could someday lead to treatments that target these diseases. But it all starts with the protein's structure, and this starts with a process in which the protein is crystallized and then analyzed using light rays brighter than the sun.

Keith Henderson helped a team of Nobel laureates unravel the structure of the LDL receptor protein at ALS beamline 8.2.1.

"We were able to see the structure of the LDL receptor's extracellular domain, and from this we can deduce its function," said Keith Henderson, a physicist in Berkeley Lab's Physical Biosciences division who helped the research team select a protein crystal most likely to yield its structural secrets.

How do you go from crystal to structure to function? The ALS, like all synchrotrons, accelerates electrons to nearly the speed of light and bends them in a circular path by powerful magnets. At this speed, electrons emit extremely bright x-ray light that is directed along a beamline toward an object that researchers want to investigate at the atomic level, such as a crystallized protein.

The pattern in which x-rays diffract off of the crystal reveals the protein's molecular structure, which reveals its function. In this case, the ALS helped determine precisely how the LDL receptor captures cholesterol, pulls it inside a cell where it is released and metabolized, and then cycles back to the cell surface to grab more cholesterol.

Such success requires painstaking preparation. First, the LDL receptor must be crystallized in just the right way, so it best diffracts x-rays. This involves a process called crystal screening, in which a slew of promising protein crystals are evaluated to determine which has the best diffraction characteristics. In this research, the University of Texas Southwestern Medical Center team sent Henderson 60 crystals, which he analyzed using a beamline that can be tuned to resonate at several frequencies.

This important step enables the beamline to be trained on an element that is specially introduced into each protein crystal. This element, a so-called anomalous scatterer, allows the collection of several data sets, each representing a unique diffraction pattern obtained as the beamline is tuned to various wavelengths around the element's x-ray absorption edge. These data sets are combined to produce an electron density map of the crystal that offers enough detail to piece together the protein's structure. It's an elegant way to image complex proteins, provided the element is successfully embedded into the crystal — which is where Henderson's work comes in.

"Out of the 60 crystals, we found a clear candidate, meaning we knew the anomalous scatterer had stuck and its diffraction quality was promising," Henderson said.

Then it was back to the lab, where the team spent more than a year refining the crystal's properties, carefully zeroing in on a specimen good enough to reveal its molecular framework under x-ray diffraction analysis.

Finally, once the crystal was perfected, the team determined the LDL receptor's structure using data collected during the commissioning of ALS beamline 8.2.1. This beamline, part of the sector 8 superbend beamlines funded by the Howard Hughes Medical Institute, is powered by a superconducting magnet that produces photons which are nearly twice as bright, at 1 angstrom, as photons produced by standard bend magnets. Because the average length of a protein bond is about 1.5 angstroms, or 1.5 hundred-millionths of a centimeter, these super-bright photons are excellent sources of x-rays capable of probing the intricacies of protein molecules.

Together, the carefully selected protein crystal and the state-of-the-art beamline yielded a complete data set that was poured into powerful computer reconstruction programs. The result is the first image of the LDL receptor in three dimensions.

[Click for larger image]
The structure of the cellular receptor that ensnares low-density lipoprotein.

"It's further confirmation that the Advanced Light Source is a world-class facility," Henderson said, adding that the six-year study also lends credence to time-consuming research that isn't guaranteed to pan out. "It's high-risk, high-reward science."

The payoff is a better understanding of the molecular breakdowns that lead to high cholesterol. Normally, the portion of the LDL receptor that protrudes from a cell wall, called the extracellular domain, binds with LDL in the liver, pulls its cholesterol cargo inside the cell, and metabolizes it to replenish hormones, the cell membrane, and vitamin D. But in some people the receptor's extracellular domain is somehow hobbled, allowing cholesterol to accumulate in the bloodstream and contributing to life-threatening diseases such as atherosclerosis.

A common cause of this breakdown is familial hypercholesterolemia, a hereditary disease that affects about one in 500 people. Researchers have found more than 900 LDL receptor mutations in people with the disease, but they didn't know how these mutations disrupt the receptor's function. Now, with the blueprint of the LDL receptor in hand, they understand how mutations lead to structural changes, and how structural changes lead to high cholesterol.

"Without the receptor's structure, we're left with only biochemical evidence that something is wrong — high cholesterol caused by a mutation — but we're not sure how," Henderson said. "We're now using the receptor's form to learn its function."

And with this knowledge, researchers can pursue a pharmaceutical remedy to familial hypercholesterolemia. So far, the image has solved a longstanding mystery concerning how the LDL receptor releases cholesterol inside the cell. Here's how it works: when the LDL receptor's extracellular domain grabs LDL, it pinches off from the cell surface and sinks inside the cell to form a sac-like vesicle called an endosome. Next, the endosome becomes more acidic, which triggers the receptor to discard the LDL. Once free of LDL, the receptor migrates back to the cell surface — a back-and-forth journey it repeats many times in its bid to cleanse the bloodstream of cholesterol.

The image also adds to the illustrious careers of three University of Texas Southwestern Medical Center researchers. Senior author Johann Deisenhofer, who is also a Howard Hughes Medical Institute investigator, received the 1988 Nobel Prize in chemistry for research using x-ray crystallography to reveal the three-dimensional structure of protein in cell membranes. And in 1985, Michael Brown and Joseph Goldstein shared the Nobel Prize in physiology or medicine for discoveries concerning cholesterol metabolism. The study, "Structure of the LDL receptor extracellular domain at endosomal pH," appeared in the December 20, 2002, issue of Science.