Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 reveal insight into nuclease specificity and dynamics.

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics

Modulation of the Activity of <i>Mycobacterium tuberculosis</i> LipY by Its PE Domain

<div><p><i>Mycobacterium tuberculosis</i> harbors over 160 genes encoding PE/PPE proteins, several of which have roles in the pathogen’s virulence. A number of PE/PPE proteins are secreted via Type VII secretion systems known as the ESX secretion systems. One PE protein, LipY, has a triglyceride lipase domain in addition to its PE domain. LipY can regulate intracellular triglyceride levels and is also exported to the cell wall by one of the ESX family members, ESX-5. Upon export, LipY’s PE domain is removed by proteolytic cleavage. Studies using cells and crude extracts suggest that LipY’s PE domain not only directs its secretion by ESX-5, but also functions to inhibit its enzymatic activity. Here, we attempt to further elucidate the role of LipY’s PE domain in the regulation of its enzymatic activity. First, we established an improved purification method for several LipY variants using detergent micelles. We then used enzymatic assays to confirm that the PE domain down-regulates LipY activity. The PE domain must be attached to LipY in order to effectively inhibit it. Finally, we determined that full length LipY and the mature lipase lacking the PE domain (LipYΔPE) have similar melting temperatures. Based on our improved purification strategy and activity-based approach, we concluded that LipY’s PE domain down-regulates its enzymatic activity but does not impact the thermal stability of the enzyme.</p></div

Biochemical Analysis of the Lipoprotein Lipase Truncation Variant, LPLS447X, Reveals Increased Lipoprotein Uptake

Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPLS447X, causes a gain of function. This mutation truncates two amino acids from LPL's C-terminus. Carriers of LPLS447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPLS447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPLS447X results in a serum lipid profile more favorable than that of LPL. In vitro reports vary as to whether LPLS447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPLS447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the Ki for ANGPTL4 inhibition of LPLS447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPLS447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPLS447X truncation exposes residues implicated in LPL binding to uptake receptors

LipY and LipYΔPE share similar thermal unfolding profiles.

<p>CD temperature scans of LipY (black) and LipYΔPE (gray) monitored at 222 nm between 25 and 95°C. Data points were collected every 1°C.</p

There are more active LipY molecules in peak 1 compared to peak 2.

<p>(A) Equal concentrations of <i>total</i> LipY Peak 1 and Peak 2 were incubated with a 1.5 molar excess of TAMRA-FP serine hydrolase probe for 30 minutes at room temperature. A standard was created using the probe alone. The amount of active Peak 1 and Peak 2 were calculated based on the standard curve. (B) Bar graph showing the V_max and K_m from Michaelis-Menten curves comparing equal amounts of <i>total</i> LipY from Peak 1 and Peak 2 using the DGGR substrate. Error bars represent the standard error of the mean of 6 independent measurements[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135447#pone.0135447.ref032" target="_blank">32</a>].</p

New insights into RNA processing by the eukaryotic tRNA splicing endonuclease

Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades, it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA-bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here, we review these recent discoveries, their implications, and the new questions raised by these findings

Size exclusion chromatography of LipY with or without Tween 20.

<p>(A) LipY purified via Nickel affinity chromatography was resolved on a Superdex 200 gel filtration column with (gray trace) or without (black trace) pre-incubation with a 270 fold molar excess of Tween 20. In the presence of detergent a second peak in the included volume appears whereas a single peak in the included volume is observed when no detergent is present. (B) LipY purified in the void volume (Peak 1) was pre-incubated with a 270 fold molar excess of Tween 20 and subjected to size exclusion chromatography (black trace). This trace is overlayed with a trace of 6 mM Tween 20 alone (gray trace). Western blots against the His tag of LipY were used to detect the presence of protein eluted from the gel filtration column in each experiment.</p

The PE domain contributes to LipY aggregation <i>in vitro</i>.

<p>LipYΔPE and the PE domain were resolved on a Superdex 200 gel filtration column. Both proteins were pre-incubated with a 270 fold molar excess of Tween 20 for 1 hour prior to being resolved on the column. In the gray PE domain trace, Peak 1 (~9.5 mL) elutes near the void volume and Peak 2 (~13 mL) elutes in the included volume. In the black LipYΔPE trace, a weak A₂₈₀ signal was observed between 9 and 12 mL and a single distinct peak (Peak 2) elutes in the included volume. Western blots against the His tag of each construct show that the majority of the PE domain elutes in the void volume while the majority of LipYΔPE elutes in the included volume.</p

Biochemical Analysis of the Lipoprotein Lipase Truncation Variant, LPL^S447X, Reveals Increased Lipoprotein Uptake

Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPL^S447X, causes a gain of function. This mutation truncates two amino acids from LPL’s C-terminus. Carriers of LPL^S447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPL^S447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPL^S447X results in a serum lipid profile more favorable than that of LPL. <i>In vitro</i> reports vary as to whether LPL^S447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPL^S447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the <i>K</i>_i for ANGPTL4 inhibition of LPL^S447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPL^S447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPL^S447X truncation exposes residues implicated in LPL binding to uptake receptors