Search

For decades, researchers have been determined to elucidate essential enzymatic functions on the atomic lengths scale by tracing atomic positions in real time. Our work builds on new possibilities unleashed by mix-and-inject serial crystallography (MISC) 1–5 at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals 6. Here, we report in atomic detail and with millisecond time-resolution how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating 7–9, cooperativity, induced fit 10,11 and conformational selection 11–13 all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme non-covalently before reacting to a trans-enamine. This was made possible in part by the application of the singular value decomposition 14 to the MISC data using a newly developed program that remains functional even if unit cell parameters change during the reaction.

After decades of diligent development at synchrotron light sources, the field of time-resolved crystallography (TRX) is elevating to new heights. Driven by the appearance of X-ray free electron lasers and supported by serial crystallography, a large number of biological systems can now be investigated with TRX. This short review cannot comprehensively cover all of them, but it will shine light on, and provide a connection between, some of the recent and past results.

There is an urgent need for anti-viral agents that treat and/or prevent Covid-19 caused by SARS-Coronavirus (CoV-2) infections. The replication of the SARS CoV-2 is dependent on the activity of two cysteine proteases, a papain-like protease, PL-pro, and the 3C-like protease known as main protease Mpro or 3CLpro. The shortest and the safest path to clinical use is the repurposing of drugs with binding affinity to PLpro or 3CLpro that have an established safety profile in humans. Several studies have reported crystal structures of SARS-CoV-2 main protease in complex with FDA approved drugs such as those used in treatment of hepatitis C. Here, we report the crystal structure of 3CLpro in complex Vitamin C (L-ascorbate) bound to the protein’s active site at 2.5 Ångstrom resolution. The crystal structure of the Vitamin C 3CLpro complex may aid future studies on the effect of Vitamin C not only on the coronavirus main protease but on related proteases of other infectious viruses.

18 pags, 11 figs, 5 tabs, Here, 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.

Phytochromes are red/far-red light photoreceptors in bacteria to plants, which elicit a variety of important physiological responses. They display a reversible photocycle between the resting (dark) Pr state and the light activated Pfr state, in which light signals are received and transduced as structural change through the entire protein to modulate the activity of the protein. It is unknown how the Pr-to-Pfr interconversion occurs as the structure of intermediates remain notoriously elusive. Here, we present short-lived crystal structures of the classical phytochrome from myxobacterium Stigmatella aurantiaca captured by an X-ray Free Electron Laser 5 ns and 33ms after light illumination of the Pr state. We observe large structural displacements of the covalently bound bilin chromophore, which trigger a bifurcated signaling pathway. The snapshots show with atomic precision how the signal progresses from the chromophore towards the output domains, explaining how plants, bacteria and fungi sense red light.

The SARS coronavirus 2 main protease 3CLpro tailor cuts various essential virus proteins out of long poly-protein translated from the virus RNA. If the 3CLpro is inhibited, the functional virus proteins cannot form and the virus cannot replicate and assemble. Any compound that inhibits the 3CLpro is therefore a potential drug to end the pandemic. Here we show that the diffraction power of 3CLpro crystals is effectively destroyed by Ebselen. It appears that Ebselen may be a widely available, relatively cost effective way to eliminate the SARS coronavirus 2.

With time-resolved crystallography (TRX), it is possible to follow the reaction dynamics in biological macromolecules by investigating the structure of transient states along the reaction coordinate. X-ray free electron lasers (XFELs) have enabled TRX experiments on previously uncharted femtosecond timescales. Here, we review the recent developments, opportunities, and challenges of pump-probe TRX at XFELs.