Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Biophysical characterization and a roadmap towards the NMR solution structure of G0S2, a key enzyme in non-alcoholic fatty liver disease.

In the United States non-alcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, affecting an estimated 80 to 100 million people. It occurs in every age group, but predominantly in people with risk factors such as obesity and type 2 diabetes. NAFLD is marked by fat accumulation in the liver leading to liver inflammation, which may lead to scarring and irreversible damage progressing to cirrhosis and liver failure. In animal models, genetic ablation of the protein G0S2 leads to alleviation of liver damage and insulin resistance in high fat diets. The research presented in this paper aims to aid in rational based drug design for the treatment of NAFLD by providing a pathway for a solution state NMR structure of G0S2. Here we describe the expression of G0S2 in an E. coli system from two different constructs, both of which are confirmed to be functionally active based on the ability to inhibit the activity of Adipose Triglyceride Lipase. In one of the constructs, preliminary NMR spectroscopy measurements show dominant alpha-helical characteristics as well as resonance assignments on the N-terminus of G0S2, allowing for further NMR work with this protein. Additionally, the characterization of G0S2 oligomers are outlined for both constructs, suggesting that G0S2 may defensively exist in a multimeric state to protect and potentially stabilize the small 104 amino acid protein within the cell. This information presented on the structure of G0S2 will further guide future development in the therapy for NAFLD.

Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma.

Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and ß-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.