Molecular structure could advance understanding of human disorders

August 27, 2001

EVANSTON, Ill. -- A recent breakthrough by scientists at Northwestern University could advance understanding of the biochemical causes of some nervous system disorders, including forms of amyotrophic lateral sclerosis (ALS) or Lou Gehrig's disease.

A team led by Amy Rosenzweig, assistant professor of biochemistry, molecular biology and cell biology and of chemistry, in collaboration with Thomas O'Halloran, professor of chemistry, is the first to determine the molecular structure of a metallochaperone (a protein that delivers metals to enzymes that need them to function) bound to its target protein.

Specifically, the researchers have shown how the copper metallochaperone CCS binds to its target, superoxide dismutase (SOD), an enzyme that, when in a mutated form, has been linked to an inherited form of ALS known as familial ALS (FALS).

"The findings are truly groundbreaking," said Val Culotta, professor of environmental health sciences at Johns Hopkins University. "For the first time, we have a glimpse of a docked complex between a copper enzyme and its copper chaperone partner and have gained new insight -- in remarkable detail -- into a metal transfer mechanism. Given the large number of human disorders that have been attributed to copper and oxidative stress, this study is both timely and relevant."

The results will be published in the September issue of Nature Structural Biology.

"We have the first image of the structure that delivers copper to its target," said postdoctoral fellow Audrey Lamb, the paper's lead author. "The coil or arm of the metallochaperone was known to exist but had never before been seen. Our structure shows the arm holding the door open for the copper to be deposited into the active site on the target protein."

Metallochaperones are essential for the proper functioning of cells. They deliver heavy metal ions, such as copper and iron, to enzymes that need them to catalyze vital biochemical reactions, such as cellular respiration, DNA synthesis and antioxidant defense. Knowledge of how metallochaperones recognize their targets, bind to target enzymes and deliver their cargo is key to understanding diseases related to these processes.

"SOD is an important enzyme -- it detoxifies free oxygen radicals that can damage cells," said Rosenzweig. "But it needs copper for this to happen. That's why it is so important that we understand how the enzyme gets its copper, which is available in limited quantities in the body."

The researchers studied the yeast versions of CCS and SOD, which are very similar to the forms in human cells. (The SOD form studied contains copper and zinc.) They determined the molecular structure by analyzing crystals of the CCS-SOD complex using the extremely brilliant X-rays produced by the Advanced Photon Source (APS) synchrotron at Argonne National Laboratory in Illinois.

The results suggest that the complex is formed in a series of steps. The metallochaperone begins as two copies of CCS bound together (known as a homodimer because of the like parts). Similarly, the target protein starts as two SOD copies bound together.

Then the two homodimers each break apart, splitting into unbound copies of CCS and SOD. Because of their similar shapes, CCS recognizes SOD, and they come together, almost like a handshake, to form what is called a heterodimer (one copy of CCS and one of SOD). Once joined, a long coil or arm swings down from the metallochaperone to the copper-binding site on the target protein, poised to deposit the copper.

Once the copper has been delivered (although copper was not actually used in the experiments in order to stabilize the complex) the heterodimer splits apart. Two copper-loaded SOD proteins then join together in order to execute its biochemical reaction.

Because the CCS-SOD complex is so small -- about 100,000 times smaller than a cell -- it could not be imaged directly. Instead, the researchers made the complex in solution, then created a crystal. Using the method of X-ray diffraction, they bombarded the crystal with X-rays, which bounced off the atoms within the crystal. By collecting and analyzing this information, Rosenzweig's team determined the location of each atom within the structure, thus creating a three-dimensional picture of the complex.

The high intensity X-rays at the DuPont-Northwestern-Dow beamline at the APS enabled the researchers to image the complex to a resolution of 2.9 angstroms. (An angstrom is one ten-billionth of a meter, or about one-hundred-millionth of an inch.) This resolution was critical for an accurate picture of how the 5,981 atoms in the complex are assembled.
-end-
The research was supported by the National Institute of General Medical Sciences (a component of the National Institutes of Health) and the ALS Association.

Northwestern University

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