Structural and Biochemical Characterization of Porcine Epidemic Diarrhea Virus Papain-Like Protease 2.

Coronaviral papain-like proteases (PLpros) are essential enzymes that mediate not only the proteolytic processes of viral polyproteins during virus replication but also the deubiquitination and deISGylation of cellular proteins that attenuate host innate immune responses. Therefore, PLpros are attractive targets for antiviral drug development. Here, we report the crystal structure of papain-like protease 2 (PLP2) of porcine epidemic diarrhea virus (PEDV) in complex with ubiquitin (Ub). The X-ray structural analyses reveal that PEDV PLP2 interacts with the Ub substrate mainly through the Ub core region and C-terminal tail. Mutations of Ub-interacting residues resulted in a moderately or completely abolished deubiquitinylating function of PEDV PLP2. In addition, our analyses also indicate that 2-residue-extended blocking loop 2 at the S4 subsite contributes to the substrate selectivity and binding affinity of PEDV PLP2. Furthermore, the PEDV PLP2 Glu99 residue, conserved in alphacoronavirus PLpros, was found to govern the preference of a positively charged P4 residue of peptidyl substrates. Collectively, our data provided structure-based information for the substrate binding and selectivity of PEDV PLP2. These findings may help us gain insights into the deubiquitinating (DUB) and proteolytic functions of PEDV PLP2 from a structural perspective. IMPORTANCE Current challenges in coronaviruses (CoVs) include a comprehensive understanding of the mechanistic effects of associated enzymes, including the 3C-like and papain-like proteases. We have previously reported that the PEDV PLP2 exhibits a broader substrate preference, superior DUB function, and inferior peptidase activity. However, the structural basis for these functions remains largely unclear. Here, we show the high-resolution X-ray crystal structure of PEDV PLP2 in complex with Ub. Integrated structural and biochemical analyses revealed that (i) three Ub core-interacting residues are essential for DUB function, (ii) 2-residue-elongated blocking loop 2 regulates substrate selectivity, and (iii) a conserved glutamate residue governs the substrate specificity of PEDV PLP2. Collectively, our findings provide not only structural insights into the catalytic mechanism of PEDV PLP2 but also a model for developing antiviral strategies.

Klotho Exerts an Emerging Role in Cytokinesis.

The Klotho gene functions as an anti-aging gene. A previous klotho-knockout mice study indicated that neither male nor female gametocytes could accomplish the first meiotic division. It suggested that Klotho might regulate cell division. In this study, we determined the roles of Klotho in cytokinesis in cultural human cells (HEK293 and HeLa) and in zebrafish embryos. Immunoprecipitation, mass spectrometry analysis, and a zebrafish model were used in this study. The results showed that Klotho is located in the midbody, which correlated with cytokinesis related kinases, Aurora kinase B and citron kinases, in the late stage of cytokinesis. There was a spatial correlation between the abscission site and the location of Klotho in the cytokinesis bridge. A three-dimensional structural reconstruction study demonstrated there was a spatial correlation among Klotho, Aurora kinase B, and citron kinases in the midbody. In addition, Klotho depletion inactivated Aurora kinases; it was also indicated that Klotho depletion caused aberrant cell cycle and delayed cytokinesis in a cell model. The study with zebrafish embryos suggested that klotho knockdown caused early embryo development abnormality due to dysregulated cytokinesis. In conclusion, Klotho might have a critical role in cytokinesis regulation by interacting with the cytokinesis related kinases.

Porcine epidemic diarrhea virus papain-like protease 2 can be noncompetitively inhibited by 6-thioguanine.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus (CoV) discovered in the 1970s that infects the intestinal tract of pigs, resulting in diarrhea and vomiting. It can cause extreme dehydration and death in neonatal piglets. In Asia, modified live attenuated vaccines have been used to control PEDV infection in recent years. However, a new strain of PEDV that belongs to genogroup 2a appeared in the USA in 2013 and then quickly spread to Canada and Mexico as well as Asian and European countries. Due to the less effective protective immunity provided by the vaccines against this new strain, it has caused considerable agricultural and economic loss worldwide. The emergence of this new strain increases the importance of understanding PEDV as well as strategies for inhibiting it. Coronaviral proteases, including main proteases and papain-like proteases, are ideal antiviral targets because of their essential roles in viral maturation. Here we provide a first description of the expression, purification and structural characteristics of recombinant PEDV papain-like protease 2, moreover present our finding that 6-thioguanine, a chemotherapeutic drug, in contrast to its competitive inhibition on SARS- and MERS-CoV papain-like proteases, is a noncompetitive inhibitor of PEDV papain-like protease 2.

6-Thioguanine is a noncompetitive and slow binding inhibitor of human deubiquitinating protease USP2.

Ubiquitin-specific protease 2 (USP2) belongs to the family of deubiquitinases that can rescue protein targets from proteasomal degradation by reversing their ubiquitination. In various cancers, including prostate cancer and ovarian carcinoma, upregulation of USP2 leads to an increase in the levels of deubiquitinated substrates such as fatty acid synthase, MDM2, cyclin D1 and Aurora-A. USP2 thus plays a critical role in tumor cells' survival and therefore represents a therapeutic target. Here a leukemia drug, 6-thioguanine, was found to be a potent inhibitor of USP2. Enzyme-kinetic and X-ray crystallographic data suggest that 6-thioguanine displays a noncompetitive and slow-binding inhibitory mechanism against USP2. Our study provides a clear rationale for the clinical evaluation of 6-thioguanine for USP2-upregulated cancers.

Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in southern China in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. Ten years later, a second highly pathogenic human CoV, MERS-CoV, emerged in the Middle East and has spread to other countries in Europe, North Africa, North America and Asia. As of November 2017, MERS-CoV had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against MERS-CoV. Here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (PLpros) of MERS-CoV and SARS-CoV. Our findings suggest that disulfiram acts as an allosteric inhibitor of MERS-CoV PLpro but as a competitive (or mixed) inhibitor of SARS-CoV PLpro. The phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of SARS-CoV PLpro by disulfiram, while synergistic inhibition of MERS-CoV PLpro by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs.

The physical forces mediating self-association and phase-separation in the C-terminal domain of TDP-43.

The TAR DNA-binding protein of 43kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43's intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43's functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43's physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320-340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS.

The N-terminal domains of FLASH and Lsm11 form a 2:1 heterotrimer for histone pre-mRNA 3'-end processing.

Unlike canonical pre-mRNAs, animal replication-dependent histone pre-mRNAs lack introns and are processed at the 3'-end by a mechanism distinct from cleavage and polyadenylation. They have a 3' stem loop and histone downstream element (HDE) that are recognized by stem-loop binding protein (SLBP) and U7 snRNP, respectively. The N-terminal domain (NTD) of Lsm11, a component of U7 snRNP, interacts with FLASH NTD and these two proteins recruit the histone cleavage complex containing the CPSF-73 endonuclease for the cleavage reaction. Here, we determined crystal structures of FLASH NTD and found that it forms a coiled-coil dimer. Using solution light scattering, we characterized the stoichiometry of the FLASH NTD-Lsm11 NTD complex and found that it is a 2:1 heterotrimer, which is supported by observations from analytical ultracentrifugation and crosslinking.

A distinct holoenzyme organization for two-subunit pyruvate carboxylase.

Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the ß subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4ß4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2ß4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis.

Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.

Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.

Lys-315 at the Interfaces of Diagonal Subunits of δ-Crystallin Plays a Critical Role in the Reversibility of Folding and Subunit Assembly.

δ-Crystallin is the major structural protein in avian eye lenses and is homologous to the urea cycle enzyme argininosuccinate lyase. This protein is structurally assembled as double dimers. Lys-315 is the only residue which is arranged symmetrically at the diagonal subunit interfaces to interact with each other. This study found that wild-type protein had both dimers and monomers present in 2-4 M urea whilst only monomers of the K315A mutant were observed under the same conditions, as judged by sedimentation velocity analysis. The assembly of monomeric K315A mutant was reversible in contrast to wild-type protein. Molecular dynamics simulations showed that the dissociation of primary dimers is prior to the diagonal dimers in wild-type protein. These results suggest the critical role of Lys-315 in stabilization of the diagonal dimer structure. Guanidinium hydrochloride (GdmCl) denatured wild-type or K315A mutant protein did not fold into functional protein. However, the urea dissociated monomers of K315A mutant protein in GdmCl were reversible folding through a multiple steps mechanism as measured by tryptophan and ANS fluorescence. Two partly unfolded intermediates were detected in the pathway. Refolding of the intermediates resulted in a conformation with greater amounts of hydrophobic regions exposed which was prone to the formation of protein aggregates. The formation of aggregates was not prevented by the addition of α-crystallin. These results highlight that the conformational status of the monomers is critical for determining whether reversible oligomerization or aggregate formation occurs.

Critical Assessment of the Important Residues Involved in the Dimerization and Catalysis of MERS Coronavirus Main Protease.

BACKGROUND: A highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and other places in Saudi Arabia, and has quickly spread to European and Asian countries since September 2012. Up to the 1st October 2015 it has infected at least 1593 people with a global fatality rate of about 35%. Studies to understand the virus are necessary and urgent. In the present study, MERS-CoV main protease (Mpro) is expressed; the dimerization of the protein and its relationship to catalysis are investigated. METHODS AND RESULTS: The crystal structure of MERS-CoV Mpro indicates that it shares a similar scaffold to that of other coronaviral Mpro and consists of chymotrypsin-like domains I and II and a helical domain III of five helices. Analytical ultracentrifugation analysis demonstrated that MERS-CoV Mpro undergoes a monomer to dimer conversion in the presence of a peptide substrate. Glu169 is a key residue and plays a dual role in both dimerization and catalysis. The mutagenesis of other residues found on the dimerization interface indicate that dimerization of MERS-CoV Mpro is required for its catalytic activity. One mutation, M298R, resulted in a stable dimer with a higher level of proteolytic activity than the wild-type enzyme. CONCLUSIONS: MERS-CoV Mpro shows substrate-induced dimerization and potent proteolytic activity. A critical assessment of the residues important to these processes provides insights into the correlation between dimerization and catalysis within the coronaviral Mpro family.

Impairment of the Cellular Distribution and Stability of the Erythropoietin Receptor Through the Direct Targeting of Aristolochic Acid.

Aristolochic acid (AA) nephropathy is complicated with early onset and severe anemia. The molecular pathological mechanism of AA-induced anemia remains unclear. The aim of this study was to evaluate the putative pathological roles of the erythropoietin receptor (EPOR) in AA-induced anemia in both AA nephropathy zebrafish and cultured human renal tubular cells (HK2). Immunofluorescence staining experiments revealed that AA colocalizes with the EPOR in zebrafish embryos as well as in the cytoplasm of HK2 cells. After exogenous EPO stimulation, the EPOR was detected in the plasma membrane of HK cells. However, cotreatment with AA and EPO inhibited EPOR signaling and its membrane localization upon EPO stimulation. The results of studies with a protein synthesis inhibitor and a lysosome inhibitor revealed that AA accelerates the lysosomal degradation of EPOR. The molecular docking results suggest that AA may interact with the N-terminus of EPOR. Together with the results of light absorption and in vitro competition assays, we concluded that AA treatment impairs EPOR membrane localization, accelerates its lysosomal degradation, and consequently downregulates EPOR signaling by direct targeting. The results of this study may further detail the pathological mechanism of severe anemia complicated with AA nephropathy.

Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.

Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 Å crystal structure of the long-chain acyl-CoA carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent carboxylases. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.

Thiopurine analogs and mycophenolic acid synergistically inhibit the papain-like protease of Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a new highly pathogenic human coronaviruses that emerged in Jeddah and Saudi Arabia and has quickly spread to other countries in Middle East, Europe and North Africa since 2012. Up to 17 December 2014, it has infected at least 938 people with a fatality rate of about 36% globally. This has resulted in an urgent need to identify antiviral drugs that are active against MERS-CoV. The papain-like protease (PL(pro)) of MERS-CoV represents an important antiviral target as it is not only essential for viral maturation, but also antagonizes interferon stimulation of the host via its deubiquitination activity. Here, we report the discovery that two SARS-CoV PL(pro) inhibitors, 6-mercaptopurine (6MP) and 6-thioguanine (6TG), as well as the immunosuppressive drug mycophenolic acid, are able to inhibit MERS-CoV PL(pro). Their inhibition mechanisms and mutually binding synergistic effect were also investigated. Our results identify for the first time three inhibitors targeting MERS-CoV PL(pro) and these can now be used as lead compounds for further antiviral drug development.

Protective Role of Comfrey Leave Extracts on UV-induced Zebrafish Fin Damage.

In zebrafish, UV exposure leads to fin malformation phenotypes including fin reduction or absence. The present study evaluated UV-protective activities of comfrey leaves extracts in a zebrafish model by recording fin morphological changes. Chemopreventive effects of comfrey leave extracts were evaluated using Kaplan-Meier analysis and Cox proportional hazards regression. The results showed that (1) the mean times of return to normal fin in the UV+comfrey (50 and 100 ppm) groups were 3.43 and 2.86 days and were quicker compared with that in the UV only group (4.21 days); (2) zebrafish fins in the UV+comfrey (50 and 100 ppm) groups were 2.05 and 3.25 times more likely to return to normal than those in the UV only group; and (3) comfrey leave extracts had UV-absorbance abilities and significantly reduced ROS production in UV-exposed zebrafish embryos, which may attenuate UV-mediated apoptosis. In conclusion, comfrey leaves extracts may have the potential to be developed as UV-protective agents to protect zebrafish embryos from UV-induced damage.

Structural and functional characterization of MERS coronavirus papain-like protease.

BACKGROUNDS: A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated. RESULTS: Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference. CONCLUSIONS: Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.

Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease.

Papain-like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus (SARS-CoV). PLpro also shows significant in vitro deubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PLpro C112S mutant in complex with ubiquitin (Ub) is reported at 1.4 Šresolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4-P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PLpro-Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed an N-cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLpro inhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLpro catalysis.

Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease.

The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

Characterizing the importance of the biotin carboxylase domain dimer for Staphylococcus aureus pyruvate carboxylase catalysis.

Biotin carboxylase (BC) is a conserved component among biotin-dependent carboxylases and catalyzes the MgATP-dependent carboxylation of biotin, using bicarbonate as the CO2 donor. Studies with Escherichia coli BC have suggested long-range communication between the two active sites of a dimer, although its mechanism is not well understood. In addition, mutations in the dimer interface can produce stable monomers that are still catalytically active. A homologous dimer for the BC domain is observed in the structure of the tetrameric pyruvate carboxylase (PC) holoenzyme. We have introduced site-specific mutations into the BC domain dimer interface of Staphylococcus aureus PC (SaPC), equivalent to those used for E. coli BC, and also made chimeras replacing the SaPC BC domain with the E. coli BC subunit (EcBC chimera) or the yeast ACC BC domain (ScBC chimera). We assessed the catalytic activities of these mutants and characterized their oligomerization states by gel filtration and analytical ultracentrifugation experiments. The K442E mutant and the ScBC chimera disrupted the BC dimer and were catalytically inactive, while the F403A mutant and the EcBC chimera were still tetrameric and retained catalytic activity. The R54E mutant was also tetrameric but was catalytically inactive. Crystal structures of the R54E, F403A, and K442E mutants showed that they were tetrameric in the crystal, with conformational changes near the mutation site as well as in the tetramer organization. We have also produced the isolated BC domain of SaPC. In contrast to E. coli BC, the SaPC BC domain is monomeric in solution and catalytically inactive.