Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 NSP15 endoribonuclease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.

Ribosomal oxygenases are structurally conserved from prokaryotes to humans.

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components and in the hydroxylation of transcription factors and splicing factor proteins. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA and ribosomal proteins have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone N(ε)-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases.

Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening.

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery.

Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.

Structural basis of glycogen branching enzyme deficiency and pharmacologic rescue by rational peptide design.

Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

Lysine methylation-dependent binding of 53BP1 to the pRb tumor suppressor.

The retinoblastoma tumor suppressor protein pRb is a key regulator of cell cycle progression and mediator of the DNA damage response. Lysine methylation at K810, which occurs within a critical Cdk phosphorylation motif, holds pRb in the hypophosphorylated growth-suppressing state. We show here that methyl K810 is read by the tandem tudor domain containing tumor protein p53 binding protein 1 (53BP1). Structural elucidation of 53BP1 in complex with a methylated K810 pRb peptide emphasized the role of the 53BP1 tandem tudor domain in recognition of the methylated lysine and surrounding residues. Significantly, binding of 53BP1 to methyl K810 occurs on E2 promoter binding factor target genes and allows pRb activity to be effectively integrated with the DNA damage response. Our results widen the repertoire of cellular targets for 53BP1 and suggest a previously unidentified role for 53BP1 in regulating pRb tumor suppressor activity.

Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases.

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein "3-box" motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.

Structural basis for the regulated protease and chaperone function of DegP.

All organisms have to monitor the folding state of cellular proteins precisely. The heat-shock protein DegP is a protein quality control factor in the bacterial envelope that is involved in eliminating misfolded proteins and in the biogenesis of outer-membrane proteins. Here we describe the molecular mechanisms underlying the regulated protease and chaperone function of DegP from Escherichia coli. We show that binding of misfolded proteins transforms hexameric DegP into large, catalytically active 12-meric and 24-meric multimers. A structural analysis of these particles revealed that DegP represents a protein packaging device whose central compartment is adaptable to the size and concentration of substrate. Moreover, the inner cavity serves antagonistic functions. Whereas the encapsulation of folded protomers of outer-membrane proteins is protective and might allow safe transit through the periplasm, misfolded proteins are eliminated in the molecular reaction chamber. Oligomer reassembly and concomitant activation on substrate binding may also be critical in regulating other HtrA proteases implicated in protein-folding diseases.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming.

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)(18)-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K(528)R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

Squeezing the most from every crystal: the fine details of data collection.

Modern synchrotron beamlines offer instrumentation of unprecedented quality, which in turn encourages increasingly marginal experiments, and for these, as much as ever, the ultimate success of data collection depends on the experience, but especially the care, of the experimenter. A representative set of difficult cases has been encountered at the Structural Genomics Consortium, a worldwide structural genomics initiative of which the Oxford site currently deposits three novel human structures per month. Achieving this target relies heavily on frequent visits to the Diamond Light Source, and the variety of crystal systems still demand customized data collection, diligent checks and careful planning of each experiment. Here, an overview is presented of the techniques and procedures that have been refined over the years and that are considered synchrotron best practice.

Structure of MMACHC reveals an arginine-rich pocket and a domain-swapped dimer for its B12 processing function.

Defects in the MMACHC gene represent the most common disorder of cobalamin (Cbl) metabolism, affecting synthesis of the enzyme cofactors adenosyl-Cbl and methyl-Cbl. The encoded MMACHC protein binds intracellular Cbl derivatives with different upper axial ligands and exhibits flavin mononucleotide (FMN)-dependent decyanase activity toward cyano-Cbl as well as glutathione (GSH)-dependent dealkylase activity toward alkyl-Cbls. We determined the structure of human MMACHC·adenosyl-Cbl complex, revealing a tailor-made nitroreductase scaffold which binds adenosyl-Cbl in a "base-off, five-coordinate" configuration for catalysis. We further identified an arginine-rich pocket close to the Cbl binding site responsible for GSH binding and dealkylation activity. Mutation of these highly conserved arginines, including a replication of the prevalent MMACHC missense mutation, Arg161Gln, disrupts GSH binding and dealkylation. We further showed that two Cbl-binding monomers dimerize to mediate the reciprocal exchange of a conserved "PNRRP" loop from both subunits, serving as a protein cap for the upper axial ligand in trans and required for proper dealkylation activity. Our dimeric structure is supported by solution studies, where dimerization is triggered upon binding its substrate adenosyl-Cbl or cofactor FMN. Together our data provide a structural framework to understanding catalytic function and disease mechanism for this multifunctional enzyme.

Molecular adaptation of the DegQ protease to exert protein quality control in the bacterial cell envelope.

To react to distinct stress situations and to prevent the accumulation of misfolded proteins, all cells employ a number of proteases and chaperones, which together set up an efficient protein quality control system. The functionality of proteins in the cell envelope of Escherichia coli is monitored by the HtrA proteases DegS, DegP, and DegQ. In contrast with DegP and DegS, the structure and function of DegQ has not been addressed in detail. Here, we show that substrate binding triggers the conversion of the resting DegQ hexamer into catalytically active 12- and 24-mers. Interestingly, substrate-induced oligomer reassembly and protease activation depends on the first PDZ domain but not on the second. Therefore, the regulatory mechanism originally identified in DegP should be a common feature of HtrA proteases, most of which encompass only a single PDZ domain. Using a DegQ mutant lacking the second PDZ domain, we determined the high resolution crystal structure of a dodecameric HtrA complex. The nearly identical domain orientation of protease and PDZ domains within 12- and 24-meric HtrA complexes reveals a conserved PDZ1 → L3 → LD/L1/L2 signaling cascade, in which loop L3 senses the repositioned PDZ1 domain of higher order, substrate-engaged particles and activates protease function. Furthermore, our in vitro and in vivo data imply a pH-related function of DegQ in the bacterial cell envelope.

Experiences From Developing Software for Large X-Ray Crystallography-Driven Protein-Ligand Studies.

The throughput of macromolecular X-ray crystallography experiments has surged over the last decade. This remarkable gain in efficiency has been facilitated by increases in the availability of high-intensity X-ray beams, (ultra)fast detectors and high degrees of automation. These developments have in turn spurred the development of several dedicated centers for crystal-based fragment screening which enable the preparation and collection of hundreds of single-crystal diffraction datasets per day. Crystal structures of target proteins in complex with small-molecule ligands are of immense importance for structure-based drug design (SBDD) and their rapid turnover is a prerequisite for accelerated development cycles. While the experimental part of the process is well defined and has by now been established at several synchrotron sites, it is noticeable that software and algorithmic aspects have received far less attention, as well as the implications of new methodologies on established paradigms for structure determination, analysis, and visualization. We will review three key areas of development of large-scale protein-ligand studies. First, we will look into new software developments for batch data processing, followed by a discussion of the methodological changes in the analysis, modeling, refinement and deposition of structures for SBDD, and the changes in mindset that these new methods require, both on the side of depositors and users of macromolecular models. Finally, we will highlight key new developments for the presentation and analysis of the collections of structures that these experiments produce, and provide an outlook for future developments.

Of problems and opportunities-How to treat and how to not treat crystallographic fragment screening data.

In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.

Novel Starting Points for Human Glycolate Oxidase Inhibitors, Revealed by Crystallography-Based Fragment Screening.

Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme's substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.

Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16.

Since the emergence of SARS-CoV-2 in 2019, Covid-19 has developed into a serious threat to our health, social and economic systems. Although vaccines have been developed in a tour-de-force and are now increasingly available, repurposing of existing drugs has been less successful. There is a clear need to develop new drugs against SARS-CoV-2 that can also be used against future coronavirus infections. Non-structural protein 10 (nsp10) is a conserved stimulator of two enzymes crucial for viral replication, nsp14 and nsp16, exhibiting exoribonuclease and methyltransferase activities. Interfering with RNA proofreading or RNA cap formation represents intervention strategies to inhibit replication. We applied fragment-based screening using nano differential scanning fluorometry and X-ray crystallography to identify ligands targeting SARS-CoV-2 nsp10. We identified four fragments located in two distinct sites: one can be modelled to where it would be located in the nsp14-nsp10 complex interface and the other in the nsp16-nsp10 complex interface. Microscale thermophoresis (MST) experiments were used to quantify fragment affinities for nsp10. Additionally, we showed by MST that the interaction by nsp14 and 10 is weak and thereby that complex formation could be disrupted by small molecules. The fragments will serve as starting points for the development of more potent analogues using fragment growing techniques and structure-based drug design.

Assessment of radiation damage behaviour in a large collection of empirically optimized datasets highlights the importance of unmeasured complicating effects.

The radiation damage behaviour in 43 datasets of 34 different proteins collected over a year was examined, in order to gauge the reliability of decay metrics in practical situations, and to assess how these datasets, optimized only empirically for decay, would have benefited from the precise and automatic prediction of decay now possible with the programs RADDOSE [Murray, Garman & Ravelli (2004). J. Appl. Cryst. 37, 513-522] and BEST [Bourenkov & Popov (2010). Acta Cryst. D66, 409-419]. The results indicate that in routine practice the diffraction experiment is not yet characterized well enough to support such precise predictions, as these depend fundamentally on three interrelated variables which cannot yet be determined robustly and practically: the flux density distribution of the beam; the exact crystal volume; the sensitivity of the crystal to dose. The former two are not satisfactorily approximated from typical beamline information such as nominal beam size and transmission, or two-dimensional images of the beam and crystal; the discrepancies are particularly marked when using microfocus beams (<20 µm). Empirically monitoring decay with the dataset scaling B factor (Bourenkov & Popov, 2010) appears more robust but is complicated by anisotropic and/or low-resolution diffraction. These observations serve to delineate the challenges, scientific and logistic, that remain to be addressed if tools for managing radiation damage in practical data collection are to be conveniently robust enough to be useful in real time.

Interplay of PDZ and protease domain of DegP ensures efficient elimination of misfolded proteins.

Aberrant proteins represent an extreme hazard to cells. Therefore, molecular chaperones and proteases have to carry out protein quality control in each cellular compartment. In contrast to the ATP-dependent cytosolic proteases and chaperones, the molecular mechanisms of extracytosolic factors are largely unknown. To address this question, we studied the protease function of DegP, the central housekeeping protein in the bacterial envelope. Our data reveal that DegP processively degrades misfolded proteins into peptides of defined size by employing a molecular ruler comprised of the PDZ1 domain and the proteolytic site. Furthermore, peptide binding to the PDZ domain transforms the resting protease into its active state. This allosteric activation mechanism ensures the regulated and rapid elimination of misfolded proteins upon folding stress. In comparison to the cytosolic proteases, the regulatory features of DegP are established by entirely different mechanisms reflecting the convergent evolution of an extracytosolic housekeeping protease.

Discovery of Novel BRD4 Ligand Scaffolds by Automated Navigation of the Fragment Chemical Space.

Fragment-based drug discovery (FBDD) is a very effective hit identification method. However, the evolution of fragment hits into suitable leads remains challenging and largely artisanal. Fragment evolution is often scaffold-centric, meaning that its outcome depends crucially on the chemical structure of the starting fragment. Considering that fragment screening libraries cover only a small proportion of the corresponding chemical space, hits should be seen as probes highlighting privileged areas of the chemical space rather than actual starting points. We have developed an automated computational pipeline to mine the chemical space around any specific fragment hit, rapidly finding analogues that share a common interaction motif but are structurally novel and diverse. On a prospective application on the bromodomain-containing protein 4 (BRD4), starting from a known fragment, the platform yields active molecules with nonobvious scaffold changes. The procedure is fast and inexpensive and has the potential to uncover many hidden opportunities in FBDD.

The low-cost Shifter microscope stage transforms the speed and robustness of protein crystal harvesting.

Despite the tremendous success of X-ray cryo-crystallography in recent decades, the transfer of crystals from the drops in which they are grown to diffractometer sample mounts remains a manual process in almost all laboratories. Here, the Shifter, a motorized, interactive microscope stage that transforms the entire crystal-mounting workflow from a rate-limiting manual activity to a controllable, high-throughput semi-automated process, is described. By combining the visual acuity and fine motor skills of humans with targeted hardware and software automation, it was possible to transform the speed and robustness of crystal mounting. Control software, triggered by the operator, manoeuvres crystallization plates beneath a clear protective cover, allowing the complete removal of film seals and thereby eliminating the tedium of repetitive seal cutting. The software, either upon request or working from an imported list, controls motors to position crystal drops under a hole in the cover for human mounting at a microscope. The software automatically captures experimental annotations for uploading to the user's data repository, removing the need for manual documentation. The Shifter facilitates mounting rates of 100-240 crystals per hour in a more controlled process than manual mounting, which greatly extends the lifetime of the drops and thus allows a dramatic increase in the number of crystals retrievable from any given drop without loss of X-ray diffraction quality. In 2015, the first in a series of three Shifter devices was deployed as part of the XChem fragment-screening facility at Diamond Light Source, where they have since facilitated the mounting of over 120 000 crystals. The Shifter was engineered to have a simple design, providing a device that could be readily commercialized and widely adopted owing to its low cost. The versatile hardware design allows use beyond fragment screening and protein crystallography.