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A young scientist scores another first

Medicine@Yale, 2011 - July August


Following up on groundbreaking work of 2006, Yale structural biologist’s team strikes again, cracking enzyme akin to an Alzheimer’s culprit

“This is the material that’s not in the biochemistry textbooks yet,” says Ya Ha, Ph.D. “This is preliminary work, but it’s still a breakthrough.”

The work in question is Ha’s research team’s recent determination of the atomic structure (known in the trade as “solving” the structure) of FlaK, an enzyme found in an evolutionarily ancient microorganism native to the salt marshes of the southeastern U.S. It may seem improbable that knowing a tiny piece of this tiny creature in such intimate detail could be a biomedical breakthrough. But the research, three years in the making, marks the first time that anyone has cracked the structure of an aspartyl membrane protease, a family of enzymes of which FlaK is a member.

Moreover, FlaK has an infamous cousin—presenilin, an enzyme that plays a major role in the development of hereditary, early-onset forms of Alzheimer’s disease. Ha, associate professor of pharmacology, hopes that knowledge of FlaK’s structure will help shed light on presenilin’s form and function, which in turn could reveal new opportunities to treat or prevent Alzheimer’s in its more common forms.

“Presenilin is similar to FlaK—there’s a suggested similarity between the structures,” says Ha, who is senior author of the report of FlaK’s structure published in Nature on July 28. “We have evidence to believe that they are evolutionarily very closely related.”

This is the second time that the Ha team has solved a structure that had eluded other researchers. In a 2006 paper in Nature, Ha, then an assistant professor, and colleagues published the atomic structure of GlpG, a rhomboid protease that does its work within cell membranes. GlpG’s unusual intramembrane hydrolytic activity is similar to that of gamma-secretase, a protein complex that cleaves amyloid precursor protein (APP) into the fragments that form insoluble plaques in the brains of patients with dementia. (Ha’s lab is covering the waterfront in the Alzheimer’s realm, having also done significant structural work on APP since 2004.)

No one has yet solved the structure of presenilin, but Ha says that comparisons of FlaK’s structure with biochemical analyses of presenilin reveal three common segments that cross the cell membrane. He believes those segments are the site where APP is cleaved, the first enzymatic step down the long road that leads to Alzheimer’s disease.

Ha’s group solves structures using X-ray crystallography, the same technique Rosalind Franklin used to produce the images James Watson and Francis Crick relied on when they deciphered the double-helix structure of the DNA molecule in the 1950s.

The most painstaking part of X-ray crystallography is growing a pure crystal comprised of numerous copies of a molecule of interest, a trial-and-error process that requires great skill. For certain molecules, like the membrane proteins Ha’s group studies, this can take years. Once a suitable crystal is attained, it is mounted in an apparatus and exposed to a fine X-ray beam. Because crystals are orderly structures, the beam scatters in an orderly way. The resulting diffraction pattern, which provides an indirect glimpse of the electron density of various parts of the molecule under study, is captured by detectors for later analysis.

This procedure is repeated many times, with the crystal slightly rotated each time, until diffraction patterns have been obtained from all orientations of the crystal.

Finally, with the help of computers and whatever biochemical or other information may exist about their molecule, scientists begin the process of deriving a structure from their data. The end result depicts the position of every atom in the molecule in three dimensions.

The Ha group’s new FlaK structure, a technical tour de force, is based on X-ray crystallography data obtained at a resolution of 3.6 angstroms; a sheet of paper is about 1 million angstroms thick.

Ha is careful not to expect too much right away from the solving of FlaK’s structure, and says it will require further research to make the leap from structure to function.

“I have a blueprint of a machine, but now we need to put gas in it and take it for a test drive,” says Ha. “We’ll be able to see if it’s a car or if this is a plane that can fly.”

To do this, Ha has been working with Jonathan A. Ellman, Ph.D., Eugene Higgins Professor of Chemistry and professor of pharmacology. The two hope to gain additional structural information that will give them insights into how FlaK operates in cell membranes.

“We’re developing and designing substrate-mimicking inhibitors to try to co-crystallize with FlaK to see how they interact,” Ha explains. “This is the next step to understand how these structures work, and it will be a collaborative effort.”

Ha was one of the first new faculty members hired by Joseph Schlessinger, Ph.D., William H. Prusoff Professor of Pharmacology, when Schlessinger became chair of the Department of Pharmacology in 2001. Ha says Schlessinger’s support and patience have been essential for those in his lab to complete the long-term research projects required in structural biology.

Solving FlaK’s structure, the latest stroke of good fortune in Ha’s lab, comes at a point in his career when he has adopted a measured response to scientific success, knowing that the years of work just completed have opened up many new avenues of research yet to be begun.

“If this had happened when I was a postdoc I would have been jumping up and down,” he says. “It still excited me to be the first to produce this structure, but now I know how slow research can go—how many years it can take.”