A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins.

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A "substrate-trapping" PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.

The Scottish Structural Proteomics Facility: targets, methods and outputs.

The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.

Expression, purification, crystallization, data collection and preliminary biochemical characterization of methicillin-resistant Staphylococcus aureus Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5'-phosphate-dependent aminotransferase.

Sar2028, an aspartate/tyrosine/phenylalanine pyridoxal-5'-phosphate-dependent aminotransferase with a molecular weight of 48,168 Da, was overexpressed in methicillin-resistant Staphylococcus aureus compared with a methicillin-sensitive strain. The protein was expressed in Escherichia coli, purified and crystallized. The protein crystallized in a primitive orthorhombic Laue group with unit-cell parameters a = 83.6, b = 91.3, c = 106.0 A, alpha = beta = gamma = 90 degrees. Analysis of the systematic absences along the three principal axes indicated the space group to be P2(1)2(1)2(1). A complete data set was collected to 2.5 A resolution.

The structure of serine palmitoyltransferase; gateway to sphingolipid biosynthesis.

Sphingolipid biosynthesis commences with the condensation of L-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS). This reaction is catalysed by the PLP-dependent enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which is a membrane-bound heterodimer (SPT1/SPT2) in eukaryotes such as humans and yeast and a cytoplasmic homodimer in the Gram-negative bacterium Sphingomonas paucimobilis. Unusually, the outer membrane of S. paucimobilis contains glycosphingolipid (GSL) instead of lipopolysaccharide (LPS), and SPT catalyses the first step of the GSL biosynthetic pathway in this organism. We report here the crystal structure of the holo-form of S. paucimobilis SPT at 1.3 A resolution. The enzyme is a symmetrical homodimer with two active sites and a monomeric tertiary structure consisting of three domains. The PLP cofactor is bound covalently to a lysine residue (Lys265) as an internal aldimine/Schiff base and the active site is composed of residues from both subunits, located at the bottom of a deep cleft. Models of the human SPT1/SPT2 heterodimer were generated from the bacterial structure by bioinformatics analysis. Mutations in the human SPT1-encoding subunit have been shown to cause a neuropathological disease known as hereditary sensory and autonomic neuropathy type I (HSAN1). Our models provide an understanding of how these mutations may affect the activity of the enzyme.

Purification, crystallization and data collection of methicillin-resistant Staphylococcus aureus Sar2676, a pantothenate synthetase.

Sar2676, a pantothenate synthetase with a molecular weight of 31 419 Da from methicillin-resistant Staphylococcus aureus, has been expressed, purified and crystallized at 293 K. The protein crystallizes in a primitive triclinic lattice, with unit-cell parameters a = 45.3, b = 60.5, c = 117.6 A, alpha = 87.2, beta = 81.2, gamma = 68.4 degrees . A complete data set has been collected to 2.3 A resolution at the ESRF. Consideration of the likely solvent content suggested the asymmetric unit to contain four molecules. This has been confirmed by molecular-replacement phasing calculations, which give a solution with four monomers using a monomer of pantothenate synthetase from Escherichia coli (PDB code 1iho), which is 41% identical to Sar2676, as a search model.

Crystallization of Ranasmurfin, a blue-coloured protein from Polypedates leucomystax.

Ranasmurfin, a previously uncharacterized approximately 13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. The crystals are an intense blue colour and diffract to 1.51 A with P2(1) symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 A, beta = 93.3 degrees . Self-rotation function analysis indicates the presence of a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of the protein is related to dimer formation. Sequence data for the protein are incomplete, but thus far have identified no model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution.

Overexpression, purification, crystallization and data collection of Sulfolobus solfataricus Sso6206, a novel highly conserved protein.

Sso6206, a 10.5 kDa protein from Sulfolobus solfataricus, has been overexpressed, purified and crystallized. The protein crystallizes in space group P6(1/5)22, with unit-cell parameters a = b = 157.8, c = 307.3 A. The crystals are hexagonal bipyramids and a data set has been collected to 2.4 A resolution. Molecular replacement cannot be attempted as no convincing model can be identified. Crystals of selenomethionine-variant protein have not yet been obtained. Interestingly, crystal packing, gel filtration and mass spectrometry all suggest the native protein forms a multi-subunit oligomer consisting of >9 subunits.

Crystallization and X-ray diffraction of a halogenating enzyme, tryptophan 7-halogenase, from Pseudomonas fluorescens.

Chlorination of natural products is often required for their biological activity; notable examples include vancomycin, the last-ditch antibiotic. It is now known that many chlorinated natural products are made not by haloperoxidases, but by FADH2-dependent halogenases. The mechanism of the flavin-containing enzymes is obscure and there are no structural data. Here, crystals of PrnA (tryptophan 7-halogenase), an enzyme that regioselectively chlorinates tryptophan, cocrystallized with tryptophan and FAD are reported. The crystals belong to the tetragonal space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 67.8, c = 276.9 A. A data set to 1.8 A with 93% completeness and an Rmerge of 7.1% has been collected from a single flash-cooled crystal. A method for incorporating selenomethionine in a Pseudomonas fluorescens expression system also is reported.

Crystallization and X-ray diffraction of 5'-fluoro-5'-deoxyadenosine synthase, a fluorination enzyme from Streptomyces cattleya.

Organofluorine compounds are widely prepared throughout the chemicals industry, but their prepararion generally requires harsh fluorinating reagents and non-aqueous solvents. On the other hand, biology has hardly exploited organofluorine compounds. A very few organisms synthesize organofluorine metabolites, suggesting they have evolved a mechanism to overcome the kinetic desolvation barrier to utilizing F(-)(aq). Here, the purification and crystallization of an enzyme from Streptomyces cattleya which is responsible for the synthesis of the C-F bond during fluoroacetate and 4-fluorothreonine biosynthesis is reported. The protein crystallizes in space group C222(1), with unit-cell parameters a = 75.9, b = 130.3, c = 183.4 A, alpha = beta = gamma = 90 degrees. Data were recorded to 1.9 A at the ESRF. The structure of the protein should provide important insights into the biochemical process of C-F bond formation.