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1.
Protein & Cell ; (12): 877-888, 2021.
Article in English | WPRIM | ID: wpr-922482

ABSTRACT

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M


Subject(s)
Antiviral Agents/therapeutic use , Binding Sites , COVID-19/virology , Coronavirus Papain-Like Proteases/metabolism , Crystallography, X-Ray , Drug Evaluation, Preclinical , Drug Repositioning , High-Throughput Screening Assays/methods , Humans , Imidazoles/therapeutic use , Inhibitory Concentration 50 , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Naphthoquinones/therapeutic use , Protease Inhibitors/therapeutic use , Protein Structure, Tertiary , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification
2.
Protein & Cell ; (12): 339-351, 2020.
Article in English | WPRIM | ID: wpr-828762

ABSTRACT

Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs. Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown. We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP•BeF3-bound states. Each subunit of the hexameric ring comprises three components-the ATPase/terminase pUL15 and two regulator/fixer proteins, pUL28 and pUL33-unlike bacteriophage terminases. Distal to the nuclease domains, six ATPase domains form a central channel with conserved basic-patches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation. Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode. Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.

3.
Protein & Cell ; (12): 505-517, 2020.
Article in English | WPRIM | ID: wpr-828759

ABSTRACT

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

4.
Protein & Cell ; (12): 339-351, 2020.
Article in English | WPRIM | ID: wpr-828598

ABSTRACT

Genome packaging is a fundamental process in a viral life cycle and a prime target of antiviral drugs. Herpesviruses use an ATP-driven packaging motor/terminase complex to translocate and cleave concatemeric dsDNA into procapsids but its molecular architecture and mechanism are unknown. We report atomic structures of a herpesvirus hexameric terminase complex in both the apo and ADP•BeF3-bound states. Each subunit of the hexameric ring comprises three components-the ATPase/terminase pUL15 and two regulator/fixer proteins, pUL28 and pUL33-unlike bacteriophage terminases. Distal to the nuclease domains, six ATPase domains form a central channel with conserved basic-patches conducive to DNA binding and trans-acting arginine fingers are essential to ATP hydrolysis and sequential DNA translocation. Rearrangement of the nuclease domains mediated by regulatory domains converts DNA translocation mode to cleavage mode. Our structures favor a sequential revolution model for DNA translocation and suggest mechanisms for concerted domain rearrangements leading to DNA cleavage.

5.
Protein & Cell ; (12): 505-517, 2020.
Article in English | WPRIM | ID: wpr-828595

ABSTRACT

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

6.
Protein & Cell ; (12): 178-195, 2019.
Article in English | WPRIM | ID: wpr-757982

ABSTRACT

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. Although several HCV protease/polymerase inhibitors were recently approved by U.S. FDA, the combination of antivirals targeting multiple processes of HCV lifecycle would optimize anti-HCV therapy and against potential drug-resistance. Viral entry is an essential target step for antiviral development, but FDA-approved HCV entry inhibitor remains exclusive. Here we identify serotonin 2A receptor (5-HTR) is a HCV entry factor amendable to therapeutic intervention by a chemical biology strategy. The silencing of 5-HTR and clinically available 5-HTR antagonist suppress cell culture-derived HCV (HCVcc) in different liver cells and primary human hepatocytes at late endocytosis process. The mechanism is related to regulate the correct plasma membrane localization of claudin 1 (CLDN1). Moreover, phenoxybenzamine (PBZ), an FDA-approved 5-HTR antagonist, inhibits all major HCV genotypes in vitro and displays synergy in combination with clinical used anti-HCV drugs. The impact of PBZ on HCV genotype 2a is documented in immune-competent humanized transgenic mice. Our results not only expand the understanding of HCV entry, but also present a promising target for the invention of HCV entry inhibitor.

7.
Protein & Cell ; (12): 590-600, 2017.
Article in English | WPRIM | ID: wpr-756983

ABSTRACT

Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.


Subject(s)
Amino Acid Sequence , Animals , Antibodies, Monoclonal , Chemistry , Genetics , Metabolism , Binding Sites , Cell Line , Crystallography, X-Ray , Enterovirus A, Human , Genetics , Allergy and Immunology , Fibroblasts , Virology , Gene Expression , HEK293 Cells , Humans , Immunoglobulin Fab Fragments , Chemistry , Genetics , Metabolism , Lysosome-Associated Membrane Glycoproteins , Chemistry , Genetics , Allergy and Immunology , Mice , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Scavenger , Chemistry , Genetics , Allergy and Immunology , Receptors, Virus , Chemistry , Genetics , Allergy and Immunology , Recombinant Fusion Proteins , Chemistry , Genetics , Allergy and Immunology , Sequence Alignment , Sequence Homology, Amino Acid , Sf9 Cells , Spodoptera , Thermodynamics
8.
Protein & Cell ; (12): 516-526, 2016.
Article in English | WPRIM | ID: wpr-757409

ABSTRACT

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.


Subject(s)
Amino Acid Motifs , Binding Sites , Cell Cycle Proteins , Chemistry , Crystallography, X-Ray , Humans , Multienzyme Complexes , Chemistry , Protein Phosphatase 2 , Chemistry , Protein Structure, Quaternary , Protein-Serine-Threonine Kinases , Chemistry
9.
Protein & Cell ; (12): 562-570, 2016.
Article in English | WPRIM | ID: wpr-757402

ABSTRACT

The recent explosive outbreak of Zika virus (ZIKV) infection has been reported in South and Central America and the Caribbean. Neonatal microcephaly associated with ZIKV infection has already caused a public health emergency of international concern. No specific vaccines or drugs are currently available to treat ZIKV infection. The ZIKV helicase, which plays a pivotal role in viral RNA replication, is an attractive target for therapy. We determined the crystal structures of ZIKV helicase-ATP-Mn(2+) and ZIKV helicase-RNA. This is the first structure of any flavivirus helicase bound to ATP. Comparisons with related flavivirus helicases have shown that although the critical P-loop in the active site has variable conformations among different species, it adopts an identical mode to recognize ATP/Mn(2+). The structure of ZIKV helicase-RNA has revealed that upon RNA binding, rotations of the motor domains can cause significant conformational changes. Strikingly, although ZIKV and dengue virus (DENV) apo-helicases share conserved residues for RNA binding, their different manners of motor domain rotations result in distinct individual modes for RNA recognition. It suggests that flavivirus helicases could have evolved a conserved engine to convert chemical energy from nucleoside triphosphate to mechanical energy for RNA unwinding, but different motor domain rotations result in variable RNA recognition modes to adapt to individual viral replication.


Subject(s)
Crystallography, X-Ray , Protein Domains , RNA Helicases , Chemistry , RNA, Viral , Chemistry , Viral Proteins , Chemistry , Zika Virus
10.
Protein & Cell ; (12): 101-116, 2015.
Article in English | WPRIM | ID: wpr-757608

ABSTRACT

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le(b)) and Le(y) antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the β-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.


Subject(s)
Binding Sites , Blood Group Antigens , Chemistry , Allergy and Immunology , Caliciviridae Infections , Allergy and Immunology , Virology , Crystallography, X-Ray , Epitopes , Chemistry , Allergy and Immunology , Evolution, Molecular , Humans , Lewis Blood Group Antigens , Chemistry , Allergy and Immunology , Norovirus , Chemistry , Allergy and Immunology , Virulence , Protein Binding , Viral Proteins , Chemistry , Allergy and Immunology
11.
Protein & Cell ; (12): 351-362, 2015.
Article in English | WPRIM | ID: wpr-757579

ABSTRACT

Ebola virus (EBOV) is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA (-ssRNA) viruses, EBOV possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain (NP(core)) possesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an α-helix of EBOV NP(core) itself, which is highly conserved among filoviridae family. Combined with other biochemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.


Subject(s)
Crystallography, X-Ray , Ebolavirus , Physiology , Humans , Nucleoproteins , Chemistry , Genetics , Metabolism , Protein Structure, Tertiary , Structure-Activity Relationship , Virus Assembly , Physiology
12.
Protein & Cell ; (12): 504-517, 2015.
Article in English | WPRIM | ID: wpr-757218

ABSTRACT

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.


Subject(s)
Amino Acid Sequence , Bacterial Proteins , Chemistry , Metabolism , Catalytic Domain , Enzyme Inhibitors , Chemistry , Pharmacology , Flavonoids , Chemistry , Pharmacology , Hydro-Lyases , Chemistry , Molecular Sequence Data , Mycobacterium tuberculosis , Protein Binding , Protein Multimerization , Protein Structure, Secondary , Sequence Alignment
13.
Protein & Cell ; (12): 814-824, 2015.
Article in English | WPRIM | ID: wpr-757174

ABSTRACT

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%-90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry.


Subject(s)
Ebolavirus , Metabolism , Flow Cytometry , Glycoproteins , Metabolism , Hepatitis A Virus Cellular Receptor 1 , Hepatitis A Virus Cellular Receptor 2 , Humans , Membrane Glycoproteins , Metabolism , Membrane Proteins , Metabolism , Protein Binding , Receptors, Virus , Metabolism , Surface Plasmon Resonance , Viral Envelope Proteins , Metabolism , Viral Proteins , Metabolism
14.
Protein & Cell ; (12): 692-703, 2014.
Article in English | WPRIM | ID: wpr-757655

ABSTRACT

Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses.


Subject(s)
Acids , Chemistry , Amino Acid Sequence , Animals , Capsid Proteins , Chemistry , Genetics , Metabolism , Enterovirus A, Human , Genetics , Metabolism , Physiology , HEK293 Cells , Host-Pathogen Interactions , Humans , Hydrogen-Ion Concentration , Lysosome-Associated Membrane Glycoproteins , Chemistry , Genetics , Metabolism , Molecular Docking Simulation , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Interaction Mapping , Protein Structure, Tertiary , RNA, Viral , Genetics , Metabolism , Receptors, Scavenger , Chemistry , Genetics , Metabolism , Sequence Homology, Amino Acid , Sf9 Cells , Static Electricity , Virion , Genetics , Metabolism , Virus Attachment
15.
Protein & Cell ; (12): 548-561, 2013.
Article in English | WPRIM | ID: wpr-757780

ABSTRACT

Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.


Subject(s)
Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Humans , Malonates , Metabolism , Models, Molecular , Molecular Sequence Data , Nitrophenols , Metabolism , Organophosphorus Compounds , Metabolism , Phosphoric Monoester Hydrolases , Chemistry , Classification , Metabolism , Tartrates , Metabolism , Water , Metabolism
16.
Protein & Cell ; (12): 628-640, 2013.
Article in English | WPRIM | ID: wpr-757777

ABSTRACT

Disulfide bond-forming (Dsb) protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins. DsbA introduces disulfide bonds into folding proteins, and is re-oxidized through interaction with its redox partner DsbB. Mycobacterium tuberculosis, a Gram-positive bacterium, expresses a DsbA-like protein ( Rv2969c), an extracellular protein that has its Nterminus anchored in the cell membrane. Since Rv2969c is an essential gene, crucial for disulfide bond formation, research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection. In the present work, the crystal structures of the extracellular region of Rv2969c (Mtb DsbA) were determined in both its reduced and oxidized states. The overall structure of Mtb DsbA can be divided into two domains: a classical thioredoxin-like domain with a typical CXXC active site, and an α-helical domain. It largely resembles its Escherichia coli homologue EcDsbA, however, it possesses a truncated binding groove; in addition, its active site is surrounded by an acidic, rather than hydrophobic surface. In our oxidoreductase activity assay, Mtb DsbA exhibited a different substrate specificity when compared to EcDsbA. Moreover, structural analysis revealed a second disulfide bond in Mtb DsbA, which is rare in the previously reported DsbA structures, and is assumed to contribute to the overall stability of Mtb DsbA. To investigate the disulphide formation pathway in M.tuberculosis, we modeled Mtb Vitamin K epoxide reductase (Mtb VKOR), a binding partner of Mtb DsbA, to Mtb DsbA.


Subject(s)
Amino Acid Sequence , Bacterial Proteins , Chemistry , Metabolism , Catalytic Domain , Crystallography, X-Ray , Disulfides , Chemistry , Escherichia coli , Metabolism , Escherichia coli Proteins , Chemistry , Metabolism , Molecular Docking Simulation , Molecular Sequence Data , Mycobacterium tuberculosis , Metabolism , Oxidation-Reduction , Protein Disulfide-Isomerases , Chemistry , Metabolism , Protein Folding , Protein Structure, Tertiary , Sequence Alignment , Static Electricity
17.
Protein & Cell ; (12): 782-792, 2013.
Article in English | WPRIM | ID: wpr-757560

ABSTRACT

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported "closed" state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two "switcher" residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1', P2 and P4 are essential for substrate specificity, which was verified by our substrate binding model. In addition, we compared the in vitro cleavage efficiency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fluorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specificity. These new insights should facilitate the future rational design of efficient 2A protease inhibitors.


Subject(s)
Coxsackievirus Infections , Virology , Crystallography, X-Ray , Cysteine Endopeptidases , Chemistry , Genetics , Fluorescence Resonance Energy Transfer , Hand, Foot and Mouth Disease , Pathology , Virology , Humans , Picornaviridae , Chemistry , Genetics , Protein Conformation , Structure-Activity Relationship , Substrate Specificity , Viral Proteins , Chemistry , Genetics
18.
Protein & Cell ; (12): 903-910, 2012.
Article in English | WPRIM | ID: wpr-757849

ABSTRACT

MCP-1-induced protein-1 (MCPIP1) is a newly identified protein that is crucial to immune regulation. Mice lacking MCPIP1 gene suffer from severe immune disorders, and most of them cannot survive longer than 12 weeks. Considerable progress has been made in revealing the mechanism underlying the immune regulatory function of MCPIP1. MCPIP1 can act as an RNase to promote the mRNA degradation of some inflammatory cytokines, such as IL-6 and IL-1. Pre-microRNAs are also confirmed to be the substrate of MCPIP1 RNase. The structure of MCPIP1 N-terminal conserved domain shows a PilT N-terminus-like RNase structure, further supporting the notion that MCPIP1 has RNase activity. MCPIP1 can also deubiquitinate TNF receptor-associated factor family proteins, which are known to mediate immune and inflammatory responses. In this review, we summarize recent progress on the immune regulatory role of MCPIP1 and discuss the mechanisms underlying its function.


Subject(s)
Amino Acid Sequence , Animals , Humans , Immunity , Molecular Sequence Data , Ribonucleases , Metabolism , Transcription Factors , Chemistry , Metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins , Metabolism , Ubiquitination
19.
Protein & Cell ; (12): 308-319, 2011.
Article in English | WPRIM | ID: wpr-757101

ABSTRACT

The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.


Subject(s)
Amino Acid Sequence , Binding Sites , Catalytic Domain , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli , Guanosine Diphosphate , Metabolism , Guanosine Triphosphate , Metabolism , Humans , Models, Molecular , Molecular Conformation , Molecular Sequence Data , Plasmids , Metabolism , Protein Binding , Protein Structure, Tertiary , Genetics , Recombinant Proteins , Chemistry , Genetics , Metabolism , ral GTP-Binding Proteins , Chemistry , Genetics , Metabolism , ral Guanine Nucleotide Exchange Factor , Chemistry , Genetics , Metabolism
20.
Protein & Cell ; (12): 384-394, 2011.
Article in English | WPRIM | ID: wpr-757087

ABSTRACT

Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are a type of newly discovered antibioticresistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 months characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations. The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics, allowing the bacteria to escape the action of antibiotics. Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues, the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified. Here, we report the three-dimensional structure of NDM-1 with two catalytic zinc ions in its active site. Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity. The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-β-lactamases (MBLs) and implicate its role in wide spectrum drug resistance. We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria. Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases.


Subject(s)
Amino Acid Sequence , Anti-Bacterial Agents , Metabolism , Binding Sites , Captopril , Chemistry , Pharmacology , Catalytic Domain , Crystallography, X-Ray , Drug Resistance, Bacterial , Enterobacteriaceae , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino Acid , beta-Lactamases , Chemistry , Metabolism
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