New method reveals mechanism of key enzyme in sleeping sickness parasite

November 29, 2012 § Leave a comment

A map of intensities from almost 200,000 diffraction patterns obtained from Trypanosoma brucei cathepsin B. This map is
used to synthesize the three-dimensional structure of the enzyme.
Image courtesy of Karol Nass, Center for Free-Electron Laser Science (

A new kid on the block to determine protein structures, serial femtosecond crystallography, has proved it can hold its own against the more established X-ray crystallography. It has allowed researchers to determine how a key enzyme in the deadly sleeping sickness parasite works.

Serial femtosecond crystallography is based on X-ray free-electron lasers, which have only recently been built and made available for research, explains Henry Chapman at DESY in Germany, one of the leading authors on the Science paper that describes the work. “These machines give X-ray pulses that are about 10 billion times more intense than synchrotron sources, those large-scale facilities where most X-ray protein crystallography is done,” he says. The X-ray pulse “is so powerful that it melts through stainless steel in seconds, so at first it wasn’t clear if it would be possible to study fragile things such as proteins.”

But Chapman and his collaborators realized that because the pulses are so short, they could get a freeze-frame picture of the protein structure before the sample got vaporized. Now this led to the intriguing possibility of studying protein crystals that are too small for conventional X-ray crystallography (indeed, perhaps even ones that are too small to be seen under an optical microscope).

So when the first X-ray free-electron laser opened in 2009 at the SLAC National Accelerator Laboratory in California, the investigators joined forces with the team of Christian Betzel at the University of Hamburg in Germany to test out the idea on a cysteine protease called cathepsin B from the parasite Trypanosoma brucei, which causes sleeping sickness. The parasite causes about 30,000 deaths every year. The protease, known as TbCatB, is involved in degrading host proteins, and experiments in mice have indicated that it may be an important drug target.

The enzyme has a peptide that keeps it inactive. The mature, active enzyme comes alive when the peptide is released. Scientists have characterized the structure of the active enzyme but not that ofthe inhibited version. Chapman, Betzel and their colleagues set their sights on determining this version with serial femtosecond crystallography.

The investigators took millions of micron-sized crystals that were grown inside insect cells and fed them in a steady water spray in front of the X-ray laser. The laser fired away at the crystals like a machine gun, at a rate of 120 pulses per second. The 293,195 diffraction images collected from the firings were processed by massive parallel computing and generated a three-dimensional map of the enzyme. The enzyme’s structure was finally figured out at a resolution of 2.1 Ångström.

The structure revealed that the native, inactive precursor of the protein is glycosylated. In particular, glycosylation on the peptide that keeps the precursor inactive is key in stabilizing the inactive precursor. The investigators noted in their paper that the glycosylation on the enzyme could be exploited in developing drugs.

Betzel says that the new X-ray method has tremendous potential to change the way protein crystallography is done. It overcomes the problem of getting large enough crystals for conventional X-ray crystallography and allows structural biologists use crystals grown inside cells. The investigators are now turning their attention to other proteins that have eluded structure determination.

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