QB3 scientists pin down shape-changing enzyme

December 4, 2009
By Kaspar Mossman, QB3

QB3 researchers have peered into the fuzz of electrons in the structure of a human enzyme and found a hidden conformation that is key to the catalytic process.

Many atomic structures of proteins, including enzymes, are obtained by X-ray diffraction. UC Berkeley professor Tom Alber and graduate student James Fraser investigated how biases in the collection and interpretation of X-ray data, which consists of clouds of electron density, can conceal minor conformations essential to protein function. Their report, recently published in Nature, examines the enzyme cyclophilin A (CYPA).

The major (red) and minor (orange) conformational states essential for catalysis are identified in the electron density of a crystal of cyclophilin A.

CYPA, found in the interior of cells, is the main receptor for the immunosuppressive drug cyclosporin. Understanding how CYPA and similar enzymes work could enable scientists to design better therapeutic drugs.

Previously, nuclear magnetic resonance (NMR) experiments by Dorothee Kern and colleagues at Brandeis University showed that CYPA flips continually between "major" and "minor" structures during the reaction cycle. (The major conformation is preferred.) Because the rate of switching is similar to the rate at which CYPA produces product, some researchers believe that flipping is essential to catalysis.

But standard X-ray studies of cryogenically frozen crystals have detected only the major conformation. Fraser and Alber suspected that the ultra-low temperature might be immobilizing CYPA and skewing results. They collected X-ray data at room temperature, at which the protein can move between conformations. Then they applied a custom algorithm called “Ringer” to systematically sample the data and distinguish signal from noise.

These new processes revealed that the CYPA electron density contained a faint shadow of the minor conformation.

It was one thing to show the presence of the minor form, but another to connect it to CYPA function. To bridge this gap, the researchers created a mutant version of CYPA that greatly favored the minor conformation.  NMR measurements by Brandeis collaborators Kern and Michael Clarkson showed that the mutation created a molecular "traffic jam" within the enzyme that slowed by similar large factors both the flipping rate and the rate at which CYPA performs its job.

"This is the first time anyone has shown that a mutation that affects the catalytic rate also has a parallel effect on the dynamics” (conformational changes) of the enzyme, Fraser says.

This discovery suggests that scientists can now re-examine many other enzymes to explore how motion may be connected to function. The new structural information will expand the number of targets for therapeutic drug design.