Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment ...protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn
CaO
cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S
to S
), in which S
is the dark-stable state and S
is the last semi-stable state before O-O bond formation and O
evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S
), two-flash illuminated (2F; S
-enriched), and ammonia-bound two-flash illuminated (2F-NH
; S
-enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S
state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn
CaO
cluster in the S
and S
states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.
Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) ...domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with k
/K
= 9,600 M
s
, achieves sub-μM EC
values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.
Abstract
Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new ...antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (M
pro
also known as 3CL
pro
) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the M
pro
Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the M
pro
as potential COVID-19 therapeutics for further testing and possibly clinical trials.
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between ...pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
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•Acoustic methods inject crystal-containing droplets directly from microplate wells•On-demand acoustic injection uses crystals efficiently without orifices or clogging•Diffraction patterns from crystals measuring several tens of μm are of high quality•Complete datasets can be obtained from fewer than 50,000 crystals
Acoustic droplet ejection provides an automated tool for efficient use of protein crystals in SFX experiments. Roessler et al. used this method to deliver crystal-containing droplets into the XFEL beam to coincide with each X-ray pulse.
Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 Schneider et al. (2013). J. Phys. ...Conf. Ser.425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser.493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006. FMX, the micro‐focusing Frontier MX beamline in sector 17‐ID‐2 at NSLS‐II, covers a 5–30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s−1 at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s−1 at final storage‐ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods – serial crystallography on micrometre‐sized crystals, raster optimization of diffraction from inhomogeneous crystals, high‐resolution data collection from large‐unit‐cell crystals, room‐temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample‐screening and ligand‐binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample‐delivery methods have been implemented, such as an ultra‐high‐speed high‐precision piezo scanner goniometer Gao et al. (2018). J. Synchrotron Rad.25, 1362–1370, new microcrystal‐optimized micromesh well sample holders Guo et al. (2018). IUCrJ, 5, 238–246 and highly viscous media injectors Weierstall et al. (2014). Nat. Commun.5, 3309. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult‐to‐crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals.
The new Frontier Microfocus Macromolecular Crystallography beamline FMX in sector 17‐ID‐2 of the National Synchrotron Lightsource II provides an ultra‐bright microfocus beam and a flexible experimental station for structure determination from the most challenging crystals.
The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or M
, is a promising target for the development of novel antiviral ...therapeutics. Previous X-ray crystal structures of M
were obtained at cryogenic tem-per-ature or room tem-per-ature only. Here we report a series of high-resolution crystal structures of unliganded M
across multiple tem-per-atures from cryogenic to physiological, and another at high humidity. We inter-rogate these data sets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a perturbation-dependent conformational landscape for M
, including a mobile zinc ion inter-leaved between the catalytic dyad, mercurial conformational heterogeneity at various sites including a key substrate-binding loop, and a far-reaching intra-molecular network bridging the active site and dimer inter-face. Our results may inspire new strategies for antiviral drug development to aid preparation for future coronavirus pandemics.
structure determination from single-wavelength anomalous diffraction using native sulfur or phospho-rus in biomolecules (native-SAD) is an appealing method to mitigate the labor-intensive production ...of heavy-atom derivatives and seleno-methio-nyl substitutions. The native-SAD method is particularly attractive for membrane proteins, which are difficult to produce and often recalcitrant to grow into decent-sized crystals. Native-SAD uses lower-energy X-rays to enhance anomalous signals from sulfur or phospho-rus. However, at lower energies, the scattering and absorption of air contribute to the background noise, reduce the signals and are thus adverse to native-SAD phasing. We have previously demonstrated native-SAD phasing at an energy of 5 keV in air at the NSLS-II FMX beamline. Here, the use of a helium path developed to reduce both the noise from background scattering and the air absorption of the diffracted X-ray beam are described. The helium path was used for collection of anomalous diffraction data at 5 keV for two proteins: thaumatin and the membrane protein TehA. Although anomalous signals from each individual crystal are very weak, robust anomalous signals are obtained from data assembled from micrometre-sized crystals. The thaumatin structure was determined from 15 microcrystals and the TehA structure from 18 microcrystals. These results demonstrate the usefulness of a helium environment in support of native-SAD phasing at 5 keV.
Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a commonly used phasing method for crystal structures in the protein ...data bank. In one form it uses a protein sequence to search a structure database to find suitable templates for phasing. However, sequence information is not always available, such as when proteins are crystallized with unknown binding partner proteins or when the crystal is of a contaminant. The recent development of AlphaFold published the predicted protein structures for every protein from twenty distinct species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough to enable sequence-independent phasing of diffraction data from two crystallization contaminants of unknown sequence. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained, which allowed the identification and structure determination of YncE and YadF. Our results demonstrate the general utility of the AlphaFold-predicted structure database with respect to sequence-independent crystallographic phasing.
The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for the development of novel antiviral ...therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these data sets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a perturbation-dependent conformational landscape for Mpro, including a mobile zinc ion interleaved between the catalytic dyad, mercurial conformational heterogeneity at various sites including a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to aid preparation for future coronavirus pandemics.
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