First new biological structure solved by a free-electron laser

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A map of intensities merged using the CrystFEL software suite from almost two hundred thousand diffraction patterns obtained from in vivo grown crystals of Trypanosoma brucei cathepsin B. This map is used to synthesize the three-dimensional molecular structure of the enzyme. Image courtesy of Karol Nass, CFEL.

Hamburg, 29 November 2012. Using the world's most powerful X-ray laser, scientists have exposed a possible Achilles' heel of the sleeping sickness parasite that threatens more than 60 million people in sub-Saharan Africa. The sophisticated analysis revealed the blueprint for a molecular plug that can selectively block a vital enzyme of the parasite Trypanosoma brucei. Plugging such a tailor-made molecule into the right place of the enzyme would render it inactive, thereby killing the parasite. The team led by DESY scientist Prof. Henry Chapman from the Center of Free-Electron Laser Science (CFEL), Prof. Christian Betzel from the University of Hamburg and Dr. Lars Redecke from the joint Junior Research Group "Structural Infection Biology using new Radiation Sources (SIAS)" of the Universities of Hamburg and Lübeck report their findings in the journal "Science". "This is the first new biological structure solved with a free-electron laser," said Chapman.

The researchers had investigated tiny crystals of the parasite's enzyme cathepsin B with intense X-rays from the free-electron laser Linac Coherent Light Source (LCLS) at the US National Accelerator Laboratory SLAC in California. "The enzyme had emerged as a promising drug target in earlier trials", said Redecke, one of the first authors of the scientific paper. "The knockdown of this essential enzyme in the parasite did cure the infection in mice."

But the same enzyme is also part of the human - and in fact of all mammalian - biochemistry, and blocking it has severe consequences. With their analysis the scientists could now pinpoint distinctive structural differences between the human and the parasite's form of the enzyme. "This should in principle allow for designing a molecule that selectively blocks the parasite's enzyme while leaving the patient's intact", explained the other first author of the paper, Karol Nass, PhD student at the Hamburg School for Structure and Dynamics in Infection (SDI), funded by the Excellence Initiative of the German Federal State of Hamburg (LEXI), The researchers stress that while the finding raises hopes, a possible new drug is still a long way to go.

Fatal without treatment

Sleeping sickness, or human African trypanosomiasis (HAT), is transmitted by the bite of the tsetse fly. The Trypanosoma parasites invade the central nervous system, and without treatment the infection is usually fatal. The disease occurs in 36 sub-Saharan countries and affects mostly poor populations living in remote rural areas. Thanks to intensified control measures the number of reported cases fell steeply in recent years, but there are still millions at risk. Current treatments of HAT rely on anti-parasitic drugs developed without knowledge of the biochemical pathways. They are not always as effective and as safe as desired, and the parasites are increasingly becoming resistant to these drugs. New drugs that selectively kill the parasite without affecting the patient's own organism would be of great use.

With cathepsin B the scientists applied a novel approach by investigating tiny crystals of the enzyme that were grown in insect cells in vivo. This way the enzyme was frozen in its natural configuration that includes a native inhibitor. Because cathepsin B works as a sort of molecular scissors cutting away at other proteins, it is produced by the cell in an inhibited form and only activated when needed. In the inhibited form a small peptide molecule is blocking the cutting edge of the molecular scissors. "With the peptide still in place we could peer below a previously impenetrable part of the cathepsin B structure ", explained Betzel. There, the analysis revealed significant differences between the peptide binding sites at the parasite's and the human form of cathepsin B. "This way, nature provided us with a blueprint of what an artificial inhibitor for the parasite's enzyme could look like." The next step would be to synthesise such a tailor-made plug and test it in the lab.

The molecular structure of the enzyme was solved to the atomic level by shooting bright X-ray flashes at the tiny cathepsin B crystals, which were only about a micron (a thousandth of a millimetre) in diameter and about ten microns long on average. Crystals scatter X-rays in a characteristic way that depends on their inner structure. From the resulting diffraction pattern the structure of the crystals can be calculated, in this case revealing the structure of the enzyme. Today, crystallography is a standard technique to analyse biomolecules. Usually, scientists use modified bacteria to produce biomolecules in large amounts and try to crystallise them into the largest possible sizes of high-quality crystals afterwards. The in vivo crystallisation pioneered at the labs of Betzel and Prof. Michael Duszenko at the University of Tübingen, who is also a member of the research team, employs living cells to produce crystals. In contrast to standard crystallisation experiments, only in vivo crystallisation yielded suitable crystals of cathepsin B in a natively inhibited form.

293,195 diffraction images recorded

But the in vivo crystals are still so small that only X-ray lasers like LCLS are bright enough to produce sufficiently detailed diffraction images. The LCLS belongs to a novel class of scientific light sources called free-electron lasers that are based on powerful particle accelerators. In these machines, electrons are accelerated to high speeds, or energies, and are then forced on a tight slalom course. In every bend each electron emits a tiny flash, and all the flashes add up to an incredibly strong X-ray pulse with laser properties, that allows to resolve structures like the natively inhibited cathepsin B molecule.

To solve the cathepsin B structure the researchers had to record hundreds of thousands of diffraction images that were painstakingly stitched together afterwards. As each crystal is destroyed when hit by the powerful X-ray flash, the team fed a stream of crystals in a watery solution through a thin nozzle into the laser path. The X-ray laser fired away at 120 shots per second, where on average only every eleventh shot actually hit a crystal. This resulted in a total of 293,195 diffraction images recorded. These could only be processed with massive parallel computing, to first generate a three-dimensional map of the entire diffracting signals of the enzyme from which an image of its structure was calculated. The final result revealed the enzyme's structure with a resolution of 2.1 Ångström (one Ångström is a tenth of a nanometre or a ten-millionth of a millimetre). "Interestingly, this discovery comes exactly at the centenary of the publication of the famous X-ray diffraction equation by William Bragg in 1912," Chapman pointed out.

The team included members from DESY, the Universities of Hamburg, Lübeck, Tübingen, Uppsala and Gothenburg, the Arizona State University, SLAC, Lawrence Livermore National Laboratory, the Max Planck institute for medical research in Heidelberg and the Max Planck advanced study group at CFEL. CFEL is a joint venture of DESY, the Max Planck society and the University of Hamburg. DESY is the leading German accelerator centre and one of the leading worldwide.


Reference
"Natively inhibited Trypanosoma brucei cathepsin B structure determined using an x-ray laser"; Lars Redecke, Karol Nass et al.; "Science", 2012 (advance online publication); DOI: 10.1126/science.1229663

Background
WHO HAT fact sheet: http://www.who.int/mediacentre/factsheets/fs259/en/

Science contacts
Prof. Henry Chapman, +49 40 8998-94155 (mobil), henry.chapman@cfel.de (Prof. Chapman will be travelling on Wednesday, 28 November)
Dr. Lars Redecke, +49 40 8998-5389, redecke@biochem.uni-luebeck.de
Prof. Christian Betzel, +49 40 8998-4744, christian.betzel@uni-hamburg.de

Media contact
DESY press officer Thomas Zoufal, +49 8998-1666, presse@desy.de

Images

Microscopic representation of a tsetse fly, the biological vector of the Trypanosoma parasite. During the feeding process, the parasite is transmitted by the fly from infected to uninfected vertebrate hosts. After uptake of the trypanosomes from an infected human, the parasite reproduces itself within the tsetse fly, representing the sexual phase of the trypanosome life cycle. After several replication cycles, the parasite is again transferred to vertebrates. Image © eye of science	Download [0 MB, 896 x 896] Cartoon representation of the molecular structure Trypanosoma brucei cathepsin B. The electron density of the inhibitory propeptide and carbohydrate chains are colored in green. This new structural information was obtained from in vivo grown crystals using an X-ray free-electron laser. Image courtesy: Karol Nass, CFEL
Download [2 MB, 1724 x 1724] An X-ray diffraction pattern of a single microcrystal of Trypanosoma brucei cathepsin B captured using a 40 femtosecond pulse from an X-ray free-electron laser. Almost 200,000 patterns like this were analyzed to obtain the molecular structure of the enzyme. Image courtesy of Karol Nass, CFEL.	Download [0 MB, 1121 x 1180] An X-ray diffraction pattern of a single microcrystal of Trypanosoma brucei cathepsin B captured using a 40 femtosecond pulse from an X-ray free-electron laser. Almost 200,000 patterns like this were analyzed to obtain the molecular structure of the enzyme. Image courtesy of Karol Nass, CFEL.
Download [0 MB, 1093 x 731] Coloured electron micrograph of the bloodstream form of the Trypanosoma brucei parasite (light bluie) that causes African trypanosomiasis (also called sleeping sickness) in humans in the presence of erythrocytes (red) and lymphocytes (yellow). After infection of the mammalian host by bite of the tsetse fly, the parasite lives within the bloodstream, before it invades the central nervous system and the brain to cause fatal effects. Image courtesy of Michael Duszenko, University of Tübingen.	Download [2 MB, 1536 x 1968] Electron micrograph of the bloodstream form of the Trypanosoma brucei parasite that causes African trypanosomiasis (also called sleeping sickness) in humans. The disease occurs primarily in sub-Saharan countries and affects mostly poor populations. Today, approximately 60 million people are at risk to be infected.    Image courtesy of Michael Duszenko, University of Tübingen.
Download [0 MB, 1024 x 882] Scanning electron micrograph of a in vivo grown Trypanosoma brucei cathepsin B microcrystal that grew in an Sf9 insect cell and was extracted. Cathepsin B is an enzyme that is essential to the Trypanosoma brucei parasite and so is a potential target to develop a cure for African sleeping sickness. The molecular structure of the enzyme was solved from almost 200,000 individual microcrystals. Image courtesy of Karol Nass, CFEL.	Download [0 MB, 671 x 669] Water jet carrying millions of cathepsin B microcrystals through the X-ray laser beam. The jet is about 4 microns thin. Image courtesy of Karol Nass, CFEL