An international collaboration led by the Deutsches Elektronen-Synchrotron (DESY; Hamburg, Germany) has conducted scientific experiments at the European XFEL x-ray laser that reveal a previously unknown structure of an enzyme responsible for antibiotics resistance.
The 3.4-km-long European XFEL is designed to deliver x-ray flashes every 220 ns. To unravel the three-dimensional (3D) structure of a biomolecule, such as an enzyme, the pulses are used to obtain flash x-ray exposures of tiny crystals grown from that biomolecule. Each exposure gives rise to a characteristic diffraction pattern on the detector. If enough such patterns are recorded from all sides of a crystal, the spatial structure of the biomolecule can be calculated. The structure of a biomolecule can reveal much about how it works.
However, every crystal can only be x-rayed once since it is vaporized by the intense flash (after it has produced a diffraction pattern). So, to build up the full 3D structure of the biomolecule, a new crystal has to be delivered into the beam in time for the next flash, by spraying it across the path of the laser in a water jet. The fastest pulse rate so far of any such x-ray laser has been 120 flashes/s. To probe biomolecules at full speed, not only the crystals must be replenished fast enough—the water jet is also vaporized by the x-rays and has to recover in time.
"We revved up the speed of the water jet carrying the samples to 100 m/s, that's about as fast as the speed record in formula 1," explains Max Wiedorn, who took care of the sample delivery together with his colleague Dominik Oberthür, both from the Center for Free-Electron Laser Science (CFEL; also in Hamburg). A specially designed nozzle made sure the high-speed jet would be stable and meet the requirements.
To record x-ray diffraction patterns at this fast rate, an international consortium led by Heinz Graafsma, who heads the detector group at DESY's photon science division and is also a professor at Mid Sweden University (Sundsvall, Sweden), designed and built a custom x-ray camera for the European XFEL. The Adaptive Gain Integrating Pixel Detector (AGIPD) can not only record images as fast as the x-ray pulses arrive, it can also tune the sensitivity of every pixel individually, making the most of the delicate diffraction patterns in which the information on the structure of the sample is encoded.
The scientists first determined the structure of a very well-known sample, the enzyme lysozyme from egg white, as a touchstone to verify the system worked as expected. Indeed, the structure derived at the European XFEL perfectly matches the known lysozyme structure, showing details as fine as 0.18 nm.
As their second target, the team chose a bacterial enzyme that plays an important role in antibiotics resistance. The molecule designated CTX-M-14 β-lactamase was isolated from the bacterium Klebsiella pneumoniae whose multidrug-resistant strains are a grave concern in hospitals worldwide. Two years ago, even a pandrug-resistant strain of Klebsiella pneumoniae was identified in the U.S., according to the Centers for Disease Control and Prevention (CDC), unaffected by all 26 commonly available antibiotics.
The bacterium's enzyme CTX-M-14 β-lactamase is present in all strains. It works like a molecular pair of scissors cutting lactam rings of penicillin derived antibiotics open, thereby rendering them useless. To avoid this, antibiotics are often administered together with a compound called avibactam that blocks the molecular scissors of the enzyme. Unfortunately, mutations change the form of the scissors. "Some hospital strains of Klebsiella pneumoniae are already able to cleave even specifically developed third generation antibiotics," explains Christian Betzel, co-author of the paper and also a professor at the University of Hamburg. "If we understand how this happens, it might help to design antibiotics that avoid this problem."
The scientists investigated a complex of CTX-M-14 β-lactamase from the non-resistant, wild type of the bacterium with avibactam bound to the enzyme's active center. "The results show with 0.17 nm precision how avibactam fits snug into a sort of canyon on the enzyme's surface that marks its active center," says Markus Perbandt from the University of Hamburg, also a co-author of the paper.
The measurements show that it is possible to record high-quality structural information, which is the first step towards recording snapshots of the biochemical reaction between enzymes and their substrates at different stages with the European XFEL. Together with the research groups of co-authors Martin Aepfelbacher and Holger Rohde, professors at the University Hospital UKE in Hamburg, the team plans to use the x-ray laser as a camera to assemble those snapshots into movies of the molecular dynamics of avibactam and this β-lactamase. "Such movies would give us crucial insights into the biochemical process that could one day help us to design better inhibitors, reducing antibiotics resistance," Betzel says.
Full details of the work appear in the journal http://dx.doi.org/10.1038/s41467-018-06156-7 Nature Communications.