The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of roomtemperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å , demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.

How changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question. Here, we use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL), ambient-temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent catalysis modulates isocyanide hydratase (ICH) conformational dynamics throughout its catalytic cycle. We visualize this previously hypothetical reaction mechanism, directly observing formation of a thioimidate covalent intermediate in ICH microcrystals during catalysis. ICH exhibits a concerted helical displacement upon active-site cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained Ile152 residue. These catalysis-activated motions permit water entry into the ICH active site for intermediate hydrolysis. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady-state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. These results demonstrate that MISC can capture otherwise elusive aspects of enzyme mechanism and dynamics in microcrystalline samples, resolving long-standing questions about the connection between nonequilibrium protein motions and enzyme catalysis.

A fixed-target approach to high-throughput room-temperature serial synchrotron crystallography with oscillation is described. Patterned silicon chips with microwells provide high crystal-loading density with an extremely high hit rate. The microfocus, undulator-fed beamline at CHESS, which has compound refractive optics and a fast-framing detector, was built and optimized for this experiment. The high-throughput oscillation method described here collects 1–5° of data per crystal at room temperature with fast (10° s−1) oscillation rates and translation times, giving a crystal-data collection rate of 2.5 Hz. Partial datasets collected by the oscillation method at a storage-ring source provide more complete data per crystal than still images, dramatically lowering the total number of crystals needed for a complete dataset suitable for structure solution and refinement – up to two orders of magnitude fewer being required. Thus, this method is particularly well suited to instances where crystal quantities are low. It is demonstrated, through comparison of first and last oscillation images of two systems, that dose and the effects of radiation damage can be minimized through fast rotation and low angular sweeps for each crystal.

The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

Post-translational modification of cysteine residues can regulate protein function and is essential for catalysis by cysteine-dependent enzymes. Covalent modifications neutralize charge on the reactive cysteine thiolate anion and thus alter the active site electrostatic environment. Although a vast number of enzymes rely on cysteine modification for function, precisely how altered structural and electrostatic states of cysteine affect protein dynamics remains poorly understood.Here we use X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) affects the protein conformational ensemble. ICH exhibits a concerted helical displacement upon cysteine modification that is gated by changes in hydrogen bond strength between the cysteine thiolate and the backbone amide of the highly strained residue Ile152. The mobile helix samples alternative conformations in crystals exposed to synchrotron X-ray radiation due to the X-rayinduced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH). This oxidized cysteine residue resembles the proposed thioimidate intermediate in ICH catalysis. Neither cysteine modification nor helical disorder were observed in X-ray free electron laser (XFEL) diffraction data. Computer simulations confirm cysteine modification-gated helical motion and show how structural changes allosterically propagate through the ICH dimer. Mutations at a Gly residue (Gly150) that modulate helical mobility reduce the ICH catalytic rate and alter its presteady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Our results suggest that cysteine modification may be a common and likely underreported means for regulating protein conformational dynamics. Figure 2: Cys101 oxidation leads to a weakening of the Ile152-Cys101 H-bond. Electrostatic Poisson-Boltzmann surfaces (red negative, blue positive charge) calculated from the Cys101-Ile152 (A) and Cys-SOH-Ile152 (B) environments. Cysteine photooxidation neutralizes the negative charge of the sulfur atom, weakening the N-H … S hydrogen bond. (C-E) Schematic showing covalent modification of Cys101 weakens the hydrogen bond (red dotted/dashed line) to Ile152 and allows relaxation of backbone strain (curved arrow). (F) The catalytic thioimidate covalent intermediate resembles photooxidized Cys101-SOH. Both modifications neutralize negative charge on Cys Sγ.

High-pressure cryocooling has been developed as an alternative method for cryopreservation of macromolecular crystals and successfully applied for various technical and scientific studies. The method requires the preservation of crystal hydration as the crystal is pressurized with dry helium gas. Previously, crystal hydration was maintained either by coating crystals with a mineral oil or by enclosing crystals in a capillary which was filled with crystallization mother liquor. These methods are not well suited to weakly diffracting crystals because of the relatively high background scattering from the hydrating materials. Here, an alternative method of crystal hydration, called capillary shielding, is described. The specimen is kept hydrated via vapor diffusion in a shielding capillary while it is being pressure cryocooled. After cryocooling, the shielding capillary is removed to reduce background X-ray scattering. It is shown that, compared to previous crystal-hydration methods, the new hydration method produces superior crystal diffraction with little sign of crystal damage. Using the new method, a weakly diffracting protein crystal may be properly pressure cryocooled with little or no addition of external cryoprotectants, and significantly reduced background scattering can be observed from the resulting sample. Beyond the applications for macromolecular crystallography, it is shown that the method has great potential for the preparation of noncrystalline hydrated biological samples for coherent diffraction imaging with future X-ray sources.