Heterologous expression, purification and structural features of native Dictyostelium discoideum dye-decolorizing peroxidase bound to a natively incorporated heme

The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis

Ultrasound cavitation and exfoliation dynamics of 2D materials re-vealed in operando by X-ray free electron laser megahertz imaging

Ultrasonic liquid phase exfoliation is a promising method for the production of two-dimensional (2D) layered materials. A large number of studies have been made in investigating the underlying ultrasound exfoliation mechanisms. However, due to the experimental challenges for capturing the highly transient and dynamic phenomena in real-time at sub-microsecond time and micrometer length scales simultaneously, most theories reported to date still remain elusive. Here, using the ultra-short X-ray Free Electron Laser pulses (~25ps) with a unique pulse train structure, we applied MHz X-ray Microscopy and machine-learning technique to reveal unambiguously the full cycles of the ultrasound cavitation and graphite layer exfoliation dynamics with sub-microsecond and micrometer resolution. Cyclic fatigue shock wave impacts produced by ultrasound cloud implosion were identified as the dominant mechanism to deflect and exfoliate graphite layers mechanically. For the graphite flakes, exfoliation rate as high as ~5 angstroms per shock wave impact was observed. For the HOPG graphite, the highest exfoliation rate was ~0.15 angstroms per impact. These new findings are scientifically and technologically important for developing industrial upscaling strategies for ultrasonic exfoliation of 2D materials

First operation of the JUNGFRAU detector in 16-memory cell mode at European XFEL

The JUNGFRAU detector is a well-established hybrid pixel detector developed at the Paul Scherrer Institut (PSI) designed for free-electron laser (FEL) applications. JUNGFRAU features a charge-integrating dynamic gain switching architecture, with three different gain stages and 75 μm pixel pitch. It is widely used at the European X-ray Free-Electron Laser (EuXFEL), a facility which produces high brilliance X-ray pulses at MHz repetition rate in the form of bursts repeating at 10 Hz. In nominal configuration, the detector utilizes only a single memory cell and supports data acquisition up to 2 kHz. This constrains the operation of the detector to a 10 Hz frame rate when combined with the pulsed train structure of the EuXFEL. When configured in so-called burst mode, the JUNGFRAU detector can acquire a series of images into sixteen memory cells at a maximum rate of around 150 kHz. This acquisition scheme is better suited for the time structure of the X-rays as well as the pump laser pulses at the EuXFEL. To ensure confidence in the use of the burst mode at EuXFEL, a wide range of measurements have been performed to characterize the detector, especially to validate the detector alibration procedures. In particular, by analyzing the detector response to varying photon intensity (so called ‘intensity scan’), special attention was given to the characterization of the transitions between gain stages. The detector was operated in both dynamic gain switching and fixed gain modes. Results of these measurements indicate difficulties in the characterization of the detector dynamic gain switching response while operated in burst mode, while no major issues have been found with fixed gain operation. Based on this outcome, fixed gain operation mode with all the memory cells was used during two experiments at EuXFEL, namely in serial femtosecond protein crystallography and Kossel lines measurements. The positive outcome of these two experiments validates the good results previously obtained, and opens the possibility for a wider usage of the detector in burst operation mode, although compromises are needed on the dynamic range

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

18 pags, 11 figs, 5 tabsHere, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.This work was supported by the National Science Foundation Science and Technology Center 'BioXFEL' through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DESC0002164 (AO, algorithm design and development) and by the National Science Foundation under contract Nos. 1551489 (AO, underlying analytical models) and DBI-2029533 (AO, functional conformations). This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1450681 to JLO. The work was also supported by funds from the National Institutes of Health grant R01 GM117342-0404. Funding and support are also acknowledged from the National Institutes of Health grant R01 GM095583, from the Biodesign Center for Applied Structural Discovery at ASU, from National Science Foundation award No. 1565180 and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. KAZ was supported by the Cornell Molecular Biophysics Training Program (NIH T32-GM008267). This work was also supported by the Cluster of Excellence 'CUI: Advanced Imaging of Matter' of the Deutsche Forschungsgemeinschaft (DFG), EXC 2056, project ID 390715994. CFEL is supported by the Gottfried Wilhelm Leibniz Program of the DFG, the 'X-probe' project funded by the European Union 2020 Research and Innovation Program under Marie Sklodowska-Curie grant agreement 637295, the European Research Council, 'Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)', ERC-2013-SyG 609920, and the Human Frontiers Science Program grant RGP0010 2017. This work is also supported by the AXSIS project funded by the European Research Council under the European Union Seventh Framework Program (FP/2007-2013)/ERC Grant Agreement No. 609920.Peer reviewe

Structural changes in the acceptor site of photosystem II upon Ca2+^{2+}/Sr2+^{2+} exchange in the Mn4_4CaO5_5 Cluster Site and the Possible Long-Range Interactions

The Mn4CaO5 cluster site in the oxygen-evolving complex (OEC) of photosystem II (PSII) undergoes structural perturbations, such as those induced by Ca$^{2+}$/Sr$^{2+}$ exchanges or Ca/Mn removal. These changes have been known to induce long-range positive shifts (between +30 and +150 mV) in the redox potential of the primary quinone electron acceptor plastoquinone A (QA), which is located 40 Å from the OEC. To further investigate these effects, we reanalyzed the crystal structure of Sr-PSII resolved at 2.1 Å and compared it with the native Ca-PSII resolved at 1.9 Å. Here, we focus on the acceptor site and report the possible long-range interactions between the donor, Mn$_4$Ca(Sr)O$_5$ cluster, and acceptor sites

Determination of the PS I content of PS II core preparations using selective emission: a new emission of PS II at 780nm

Routinely prepared PS II core samples are often contaminated by a significant (∼ 1-5%) fraction of PS I, as well as related proteins. This contamination is of little importance in many experiments, but masks the optical behaviour of the deep red state

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

This perspective review describes emerging techniques and future opportunities for time-resolved serial femtosecond crystallography (TR-SFX) experiments using high repetition rate XFEL sources. High repetition rate sources are becoming more available with the European XFEL in operation and the recently upgraded LCLS-II will be available in the near future. One efficient use of these facilities for TR-SFX relies on pump–probe experiments using a laser to trigger a reaction of light-responsive proteins or mix-and-inject experiments for light-unresponsive proteins. With the view to widen the application of TR-SFX, the promising field of photocaged compounds is under development, which allows the very fast laser triggering of reactions that is no longer limited to naturally light-responsive samples. In addition to reaction triggering, a key concern when performing an SFX experiment is efficient sample usage, which is a main focus of new high repetition rate-compatible sample delivery methods