Diffract and destroy: 3D sleeping sickness enzyme

Trypanosomiasis X-rays

The X-ray structure of an enzyme crucial to the life cycle of the microbial parasite that causes African trypanosomiasis (sleeping sickness) in humans has been determined through an international collaboration.

A research team including scientists led by Henry Chapman from the Center of Free-Electron Laser Science (CFEL), German Electron Synchrotron (DESY), Christian Betzel from the University of Hamburg and Lars Redecke from the SIAS joint Junior Research Group at the Universities of Hamburg and Lübeck also includes researchers from the Max Planck Institute, Heidelberg, University of Gothenburg, University of Tübingen and Lawrence Livermore National Laboratory and Arizona State University scientists. They report details of the structure in a recent issue of the journal Science.

The protozoan Trypanosoma brucei causes sleeping sickness in humans and nagana in animals in Africa. It has an insect vector in the form of the tsetse fly and a mammalian host. The trypanosome undergoes complex changes during its life cycle to allow it to traverse the species gap from insect gut to mammalian bloodstream. The sleeping sickness parasite afflicts more than 60 million people in sub-Saharan Africa and kills an approximate 30000 people each year. Current drug treatments are not well tolerated, cause serious side effects and the parasites are becoming increasingly drug resistant. 

Enzyme focus

The cathepsin B protease enzyme (TbCatB) is the focus of the current study and represents an essential weapon used by T. brucei in its attack on the host and thus represents an important target for drug design. The team explains that this is the first new biological structure to be solved with a free-electron laser. The team combined in vivo crystallization and serial femtosecond crystallography to allow them to obtain a room-temperature 2.1 Å resolution structure of the fully glycosylated precursor complex of TbCatB.

"These images of an enzyme are the first results from our new 'diffract-then-destroy' snapshot X-ray laser method to show new biological structures which have not been seen before," explains ASU physicist John Spence. "The work was led by the DESY group and used the Linac Coherent Light Source at the US Department of Energy's SLAC National Accelerator Laboratory." 

Nanocrystalline analysis

"This paper is so exciting as it is based on nanocrystals grown by the groups at DESY in Hamburg and at the University of Lübeck inside living insect cells," explains team member and biochemist Petra Fromme. "This is the first novel structure determined by the new method of femtosecond crystallography. The structure may be of great importance for the development of new drugs to fight sleeping sickness, as it shows novel features of the structure of the CatB protein, a protease that is essential for the pathogenesis, including the structure of natural inhibitor peptide bound in the catalytic cleft of the enzyme."

In terms of targeting this enzyme with drugs, drug designers faced a major problem as cathepsin B enzyme is also found in all mammals, including humans. The detailaed 3D structure should allow researchers to pinpoint specific differences between the human and the parasite form of the enzyme allowing more precise targeting of the parasite enzyme and so avoiding interfering with the human enzyme and so preclude certain side effects. "The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the 'diffraction before destruction' approach of X-ray free-electron lasers from hundreds of thousands of individual microcrystals," the team says.

"The next steps are to push the technique to obtain structures from smaller and smaller crystals, perhaps all the way down to the single molecule, and to use it for time-resolved structural studies," Chapman told SpectroscopyNOW. "That is, we wish to obtain three-dimensional atomic-resolution molecular movies."