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Serial crystallography is a rapidly developing method for the determination of the structure of biomolecules at room temperature at near-atomic resolution from an ensemble of small crystals. Numerous advances in detectors, data analysis pipelines, sample delivery methods, and crystallization protocols expand the scope of structural biology to understand the fundamental processes that take place in living cells. Many experimental strategies for serial crystallography are in use, depending on the type and sizes of the crystals or other needs of the experiment. Such strategies should ideally minimize the wastage of samples or beamtime without compromising experimental goals. This paper proposes a way to optimize beamtime utilization in serial crystallography experiments that use fixed-target sample delivery methods, such as chips. The strategy involves two key steps. Firstly, a fast raster scan of the chip is performed to determine the positions of the crystals based on their diffraction. Subsequently, a rotational series is collected at each identified crystal position, covering a narrow range of chip orientations. This approach enables the exclusion of empty positions during data acquisition, resulting in significant savings in beam time utilization and a reduced volume of measured data.

To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics and the structural distributions they generate remains a key challenge. Here, using 1146 crystal structures of the main protease (M pro ) from SARS-CoV-2, we were able to infer a model of the enzyme’s structural fluctuations. M pro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the structural changes that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally correlated with the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography, in which numerous crystallographic datasets are analyzed, can reveal the structural fluctuations of protein native states and help uncover their biological function. Using the small differences across 1146 crystal structures we collected of the SARS-CoV-2 main protease, we map correlated atomic fluctuations to predict and experimentally confirm a functional allosteric network.

The main protease (M pro ) of SARS-CoV-2 is critical for viral function and a key drug target. M pro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized M pro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. M pro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein’s crystallization behavior is linked to its redox state. Oxidized M pro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of M pro and the crystallization conditions necessary to study this structurally. The main protease, a key enzyme of SARS-CoV-2, can protect itself from oxidative damage. Here, Reinke, Schubert, and colleagues used XFEL radiation to image the enzyme, revealing the disulfide and NOS/SONOS bonds that form in response to oxygen.

To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics and the structural distributions they generate remains a key challenge. Here, using 1146 crystal structures of the main protease (M ) from SARS-CoV-2, we were able to infer a model of the enzyme's structural fluctuations. M is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the structural changes that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally correlated with the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography, in which numerous crystallographic datasets are analyzed, can reveal the structural fluctuations of protein native states and help uncover their biological function.

SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, facilitating cleavage of the viral polypeptide chain, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to support coronaviruses in evading the host’s innate immune responses. We identified three phenolic compounds bound to PLpro, preventing essential molecular interactions to ISG15 by screening a natural compound library. The compounds identified by X-ray screening and complexed to PLpro demonstrate clear inhibition of PLpro in a deISGylation activity assay. Two compounds exhibit distinct antiviral activity in Vero cell line assays and one inhibited a cytopathic effect in non-cytotoxic concentration ranges. In the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections. Three natural phenolic compounds are found to bind to an allosteric site in SARS-CoV-2 papain-like protease and exhibit antiviral activity in vitro, showing potential as starting scaffolds for drug design.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (M pro ), its moderate activity in M pro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin’s efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections. Reinke et. al. show that targeting host proteins like cathepsins is a promising strategy for treating SARS-CoV-2 and other viral infections due to their mutational stability in contrast to highly mutable viral targets.

To interpret and transmit biological signals, proteins use correlated motions. Experimental determination of these dynamics with atomic resolution remains a key challenge. Here, using thousands of crystals of the main protease (Mpro) from SARS-CoV-2, we were able to infer a model of the protein's correlated motions. Mpro is regulated by concentration, becoming enzymatically active after forming a homodimer. To understand the correlated motions that enable dimerization to activate catalysis, we employed our model, predicting which regions of the dimerization domain are structurally linked to the active site. Mutations at these positions, expected to disrupt catalysis, resulted in a dramatic reduction in activity in one case, a mild effect in the second, and none in the third. Additional crystallography and biophysical experiments provide a mechanistic explanation for these results. Our work suggests that a statistical crystallography can determine protein correlated motions and rationalize their biological function.Competing Interest StatementThe authors have declared no competing interest.

The main protease (M ) of SARS-CoV-2 is critical for viral function and a key drug target. M is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized M showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. M forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized M spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of M and the crystallization conditions necessary to study this structurally.

Abstract 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 two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. 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. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2. Competing Interest Statement The authors have declared no competing interest.

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://www.rcsb.org/structure/6YNQ