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1.
Structure ; 31(1): 33-43.e5, 2023 01 05.
Article En | MEDLINE | ID: mdl-36513066

The lack of incorporating epitope information into the selection process makes the conventional antibody screening method less effective in identifying antibodies with desired functions. Here, we developed an epitope-directed antibody selection method by designing a directed library favoring the target epitope and a precise "counter" antigen for clearing irrelevant binders in the library. With this method, we successfully isolated an antibody, pF7_A5, that targets the less conserved region on the FZD2/7 CRD as designed. Guided by the structure of pF7_A5-FZD2CRD, a further round of evolution was conducted together with the "counter" antigen selection strategy, and ultimately, an FZD2-specific antibody and an FZD7-preferred antibody were obtained. Because of targeting the predefined functional site, all these antibodies exhibited the expected modulatory activity on the Wnt pathway. Together, the method developed here will be useful in antibody drug discovery, and the identified FZD antibodies will have clinical potential in FZD-related cancer therapy.


Antibodies, Monoclonal , Directed Molecular Evolution , Epitope Mapping , Epitopes , Frizzled Receptors , Wnt Signaling Pathway , Drug Discovery , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Frizzled Receptors/chemistry , Frizzled Receptors/genetics , Frizzled Receptors/immunology , Wnt Signaling Pathway/immunology , Antibodies, Monoclonal/immunology , Epitope Mapping/methods , Humans , Protein Conformation , Directed Molecular Evolution/methods
2.
Cell Discov ; 8(1): 90, 2022 Sep 09.
Article En | MEDLINE | ID: mdl-36085283

In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis.

3.
Life (Basel) ; 12(9)2022 Sep 16.
Article En | MEDLINE | ID: mdl-36143481

Intracellular fatty acid-binding proteins are evolutionarily highly conserved proteins. The major functions and responsibilities of this family are the regulation of FA uptake and intracellular transport. The structure of the H-FABP ortholog from mouse (Mus musculus) had not been revealed at the time this study was completed. Thus, further exploration of the structural properties of mouse H-FABP is expected to extend our knowledge of the model animal's molecular mechanism of H-FABP function. Here, we report the high-resolution crystal structure and the NMR characterization of mouse H-FABP. Our work discloses the unique structural features of mouse H-FABP, offering a structural basis for the further development of small-molecule inhibitors for H-FABP.

4.
Genomics Proteomics Bioinformatics ; 20(4): 765-779, 2022 Aug.
Article En | MEDLINE | ID: mdl-35288344

Uncovering conserved 3D protein-ligand binding patterns on the basis of functional groups (FGs) shared by a variety of small molecules can greatly expand our knowledge of protein-ligand interactions. Despite that conserved binding patterns for a few commonly used FGs have been reported in the literature, large-scale identification and evaluation of FG-based 3D binding motifs are still lacking. Here, we propose a computational method, Automatic FG-based Three-dimensional Motif Extractor (AFTME), for automatic mapping of 3D motifs to different FGs of a specific ligand. Applying our method to 233 naturally-occurring ligands, we define 481 FG-binding motifs that are highly conserved across different ligand-binding pockets. Systematic analysis further reveals four main classes of binding motifs corresponding to distinct sets of FGs. Combinations of FG-binding motifs facilitate the binding of proteins to a wide spectrum of ligands with various binding affinities. Finally, we show that our FG-motif map can be used to nominate FGs that potentially bind to specific drug targets, thus providing useful insights and guidance for rational design of small-molecule drugs.


Proteins , Ligands , Proteins/metabolism , Protein Binding , Binding Sites
5.
Nat Commun ; 13(1): 454, 2022 01 24.
Article En | MEDLINE | ID: mdl-35075127

Melatonin receptors (MT1 and MT2 in humans) are family A G protein-coupled receptors that respond to the neurohormone melatonin to regulate circadian rhythm and sleep. Numerous efforts have been made to develop drugs targeting melatonin receptors for the treatment of insomnia, circadian rhythm disorder, and cancer. However, designing subtype-selective melatonergic drugs remains challenging. Here, we report the cryo-EM structures of the MT1-Gi signaling complex with 2-iodomelatonin and ramelteon and the MT2-Gi signaling complex with ramelteon. These structures, together with the reported functional data, reveal that although MT1 and MT2 possess highly similar orthosteric ligand-binding pockets, they also display distinctive features that could be targeted to design subtype-selective drugs. The unique structural motifs in MT1 and MT2 mediate structural rearrangements with a particularly wide opening on the cytoplasmic side. Gi is engaged in the receptor core shared by MT1 and MT2 and presents a conformation deviating from those in other Gi complexes. Together, our results provide new clues for designing melatonergic drugs and further insights into understanding the G protein coupling mechanism.


Receptor, Melatonin, MT1/chemistry , Receptor, Melatonin, MT2/chemistry , Amino Acid Motifs , Cryoelectron Microscopy , Humans , Indenes/chemistry , Indenes/metabolism , Ligands , Melatonin/analogs & derivatives , Melatonin/chemistry , Melatonin/metabolism , Protein Binding , Protein Conformation , Receptor, Melatonin, MT1/genetics , Receptor, Melatonin, MT1/metabolism , Receptor, Melatonin, MT2/genetics , Receptor, Melatonin, MT2/metabolism
6.
Sci Rep ; 11(1): 11663, 2021 06 03.
Article En | MEDLINE | ID: mdl-34083615

The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.Trial registration: Clinicaltrials.gov NCT01588132.


Blood Platelets/drug effects , Blood Platelets/metabolism , Crotalid Venoms/therapeutic use , Fibrinolytic Agents/therapeutic use , Lectins, C-Type/therapeutic use , Platelet Glycoprotein GPIb-IX Complex/antagonists & inhibitors , Snake Venoms/therapeutic use , Animals , Blood Coagulation/drug effects , Crotalid Venoms/chemistry , Crotalid Venoms/isolation & purification , Crotalid Venoms/pharmacokinetics , Crotalinae , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/isolation & purification , Fibrinolytic Agents/pharmacokinetics , Healthy Volunteers , Humans , Lectins, C-Type/chemistry , Lectins, C-Type/isolation & purification , Models, Molecular , Platelet Adhesiveness/drug effects , Platelet Aggregation/drug effects , Platelet Count , Platelet Glycoprotein GPIb-IX Complex/chemistry , Protein Binding , Protein Conformation , Ristocetin/pharmacology , Snake Venoms/chemistry , Snake Venoms/isolation & purification , Snake Venoms/pharmacokinetics , Structure-Activity Relationship , Thrombin/pharmacology , Thrombosis/prevention & control , von Willebrand Factor/chemistry , von Willebrand Factor/metabolism
7.
FEBS J ; 288(20): 6019-6034, 2021 10.
Article En | MEDLINE | ID: mdl-33955674

Staphylococcus aureus is a well-known clinical pathogenic bacterium. In recent years, due to the emergence of multiple drug-resistant strains of S. aureus in clinical practice, S. aureus infections have become an increasingly severe clinical problem. Ntdp (nucleoside tri- and diphosphatase, also known as Sa1684) is a nucleotide phosphatase that has a significant effect on the proliferation of S. aureus colonies and the killing ability of the host. Here, we identified the nucleoside tri- and diphosphate hydrolysis activity of Ntdp and obtained the three-dimensional structures of apo-Ntdp and three substrate analog (ATPγ S, GDPß S, and GTPγ S) complexes of Ntdp. Through structural analysis and biochemical verification, we illustrated the structural basis for the divalent cation selectivity, substrate recognition model, and catalytic mechanism of Ntdp. We also revealed a possible basal functional pattern of the DUF402 domain and hypothesized the potential pathways by which the protein regulates the expression of the two-component regulatory factor agr and the downstream virulence factors. Overall, the above findings provide crucial insights into our understanding of the Ntdp functional mechanism in the infection process.


Bacterial Proteins/metabolism , Diphosphates/metabolism , Nucleosides/metabolism , Polyphosphates/metabolism , Staphylococcus aureus/physiology , Trans-Activators/metabolism , Virulence Factors/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Hydrolysis , Trans-Activators/chemistry , Trans-Activators/genetics , Virulence Factors/chemistry , Virulence Factors/genetics
8.
Proc Natl Acad Sci U S A ; 117(48): 30433-30440, 2020 12 01.
Article En | MEDLINE | ID: mdl-33199635

Two-component systems (TCS), which typically consist of a membrane-embedded histidine kinase and a cytoplasmic response regulator, are the dominant signaling proteins for transduction of environmental stimuli into cellular response pathways in prokaryotic cells. HptRSA is a recently identified TCS consisting of the G6P-associated sensor protein (HptA), transmembrane histidine kinase (HptS), and cytoplasmic effector (HptR). HptRSA mediates glucose-6-phosphate (G6P) uptake to support Staphylococcus aureus growth and multiplication within various host cells. How the mechanism by which HptRSA perceives G6P and triggers a downstream response has remained elusive. Here, we solved the HptA structures in apo and G6P-bound states. G6P binding in the cleft between two HptA domains caused a conformational closing movement. The solved structures of HptA in complex with the periplasmic domain of HptS showed that HptA interacts with HptS through both constitutive and switchable interfaces. The G6P-free form of HptA binds to the membrane-distal side of the HptS periplasmic domain (HptSp), resulting in a parallel conformation of the HptSp protomer pair. However, once HptA associates with G6P, its intramolecular domain closure switches the HptA-HptSp contact region into the membrane-proximal domain, which causes rotation and closure of the C termini of each HptSp protomer. Through biochemical and growth assays of HptA and HptS mutant variants, we proposed a distinct mechanism of interface switch-mediated signaling transduction. Our results provide mechanistic insights into bacterial nutrient sensing and expand our understanding of the activation modes by which TCS communicates external signals.


Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Histidine Kinase/chemistry , Histidine Kinase/metabolism , Signal Transduction , Bacterial Physiological Phenomena , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Structure-Activity Relationship
9.
J Biol Chem ; 295(30): 10293-10306, 2020 07 24.
Article En | MEDLINE | ID: mdl-32499370

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. ßγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of ßγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of ßγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.


Amphibian Proteins/chemistry , Disulfides/chemistry , Pore Forming Cytotoxic Proteins/chemistry , Protein Multimerization , Animals , Anura , Oxidation-Reduction , Protein Domains
10.
J Biol Chem ; 295(51): 17865-17876, 2020 12 18.
Article En | MEDLINE | ID: mdl-33454020

Investigations of bacterial resistance strategies can aid in the development of new antimicrobial drugs as a countermeasure to the increasing worldwide prevalence of bacterial antibiotic resistance. One such strategy involves the TipA class of transcription factors, which constitute minimal autoregulated multidrug resistance (MDR) systems against diverse antibiotics. However, we have insufficient information regarding how antibiotic binding induces transcriptional activation to design molecules that could interfere with this process. To learn more, we determined the crystal structure of SkgA from Caulobacter crescentus as a representative TipA protein. We identified an unexpected spatial orientation and location of the antibiotic-binding TipAS effector domain in the apo state. We observed that the α6-α7 region of the TipAS domain, which is canonically responsible for forming the lid of antibiotic-binding cleft to tightly enclose the bound antibiotic, is involved in the dimeric interface and stabilized via interaction with the DNA-binding domain in the apo state. Further structural and biochemical analyses demonstrated that the unliganded TipAS domain sterically hinders promoter DNA binding but undergoes a remarkable conformational shift upon antibiotic binding to release this autoinhibition via a switch of its α6-α7 region. Hence, the promoters for MDR genes including tipA and RNA polymerases become available for transcription, enabling efficient antibiotic resistance. These insights into the molecular mechanism of activation of TipA proteins advance our understanding of TipA proteins, as well as bacterial MDR systems, and may provide important clues to block bacterial resistance.


Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Trans-Activators/metabolism , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Caulobacter crescentus/metabolism , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , Dimerization , Drug Resistance, Multiple, Bacterial/genetics , Kinetics , Promoter Regions, Genetic , Protein Binding , Protein Conformation, alpha-Helical , Protein Structure, Tertiary , Sequence Alignment , Trans-Activators/antagonists & inhibitors , Trans-Activators/chemistry , Transcriptional Activation/drug effects
11.
FEBS Lett ; 594(4): 740-750, 2020 02.
Article En | MEDLINE | ID: mdl-31675429

Cyclobutane pyrimidine dimers (CPD), as a common DNA damage caused by UV radiation, often lead to skin cancer. Here, we identified a photolyase from the alga Arthrospira platensis (designated as Ap-phr), which has been regarded as a safe organism for humans for centuries, that can efficiently repair CPD lesions in ssDNA and dsDNA in vitro. The 1.6 Å resolution crystal structure of Ap-phr revealed that it possesses a unique methenyltetrahydrofolate chromophore-binding pattern with high energy transfer efficiency. Our study of Ap-phr highlights its potential use in cosmetic, industrial and aesthetic medicine applications.


Deoxyribodipyrimidine Photo-Lyase/metabolism , Spirulina/enzymology , Tetrahydrofolates/metabolism , DNA, Single-Stranded/metabolism , Deoxyribodipyrimidine Photo-Lyase/chemistry , Models, Molecular , Phylogeny , Protein Conformation
12.
FEBS J ; 287(11): 2292-2311, 2020 06.
Article En | MEDLINE | ID: mdl-31725950

The eubacterial ß sliding clamp (DnaN) plays a crucial role in DNA metabolism through direct interactions with DNA, polymerases, and a variety of protein factors. A canonical protein-DnaN interaction has been identified in Escherichia coli and some other species, during which protein partners are tethered into the conserved canonical hydrophobic crevice of DnaN via the consensus ß-binding motif. Caulobacter crescentus is an excellent research model for use in the investigation of DNA replication and cell-cycle regulation due to its unique asymmetric cell division pattern with restricted replication initiation; however, little is known about the specific features of C. crescentus DnaN (CcDnaN). Here, we report a significant divergence in the association of CcDnaN with proteins based on docking analysis and crystal structures that show that the ß-binding motifs of its protein partners bind a novel pocket instead of the canonical site. Pull-down and isothermal titration calorimetry results revealed that mutations within the novel pocket disrupt protein-CcDnaN interactions. It was also shown by replication and regulatory inactivation of DnaA assays that mediation of protein interaction by the novel pocket is closely related to the performance of CcDnaN during replication and the DnaN-mediated regulation process. Moreover, assessments of clamp competition showed that DNA does not compete with protein partners when binding to the novel pocket. Overall, our structural and biochemical analyses provide strong evidence that CcDnaN employs a noncanonical protein association pattern.


Caulobacter crescentus/genetics , DNA Replication/genetics , DNA-Binding Proteins/genetics , Transcription, Genetic , Caulobacter crescentus/growth & development , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/genetics
13.
J Struct Biol ; 208(3): 107388, 2019 12 01.
Article En | MEDLINE | ID: mdl-31520693

ArlRS is an essential two-component system in Staphylococcus aureus that regulates the transcription of virulence factors and participate in numerous pathogenic and symbiotic processes. In this work, we identified different DNA binding properties and oligomerization states among the DNA-binding domain of ArlR (ArlRDBD) and the phosphorylated and unphosphorylated full-length ArlR. Based on a 2.5-Å resolution crystal structure of ArlRDBD and subsequent mutagenesis experiments, we confirmed the DNA-binding site of ArlR and the preferred binding sequences in the agr promoter that enables the DNA recognition process. Finally, we propose a putative transcription regulation mechanism for ArlR. This work will facilitate our understanding of the DNA binding affinity regulatory mechanism between the phosphorylated and unphosphorylated response regulator in the two-component system.

14.
Cell Res ; 29(7): 562-578, 2019 07.
Article En | MEDLINE | ID: mdl-31201382

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, powered congression of those chromosomes, their segregation in anaphase, and assembly of a spindle midzone at mitotic exit. The centromere-associated kinesin motor CENP-E, whose binding partner is BubR1, has been implicated in congression of misaligned chromosomes and the transition from lateral kinetochore-microtubule association to end-on capture. Although previously proposed to be a pseudokinase, here we report the structure of the kinase domain of Drosophila melanogaster BubR1, revealing its folding into a conformation predicted to be catalytically active. BubR1 is shown to be a bona fide kinase whose phosphorylation of CENP-E switches it from a laterally attached microtubule motor to a plus-end microtubule tip tracker. Computational modeling is used to identify bubristatin as a selective BubR1 kinase antagonist that targets the αN1 helix of N-terminal extension and αC helix of the BubR1 kinase domain. Inhibition of CENP-E phosphorylation is shown to prevent proper microtubule capture at kinetochores and, surprisingly, proper assembly of the central spindle at mitotic exit. Thus, BubR1-mediated CENP-E phosphorylation produces a temporal switch that enables transition from lateral to end-on microtubule capture and organization of microtubules into stable midzone arrays.


Cell Cycle Proteins , Drosophila Proteins , Drosophila melanogaster/metabolism , Microtubules/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases , Spindle Apparatus/metabolism , Animals , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/physiology , Cloning, Molecular , Drosophila Proteins/chemistry , Drosophila Proteins/physiology , HEK293 Cells , HeLa Cells , Humans , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/physiology , Sf9 Cells
15.
J Mol Biol ; 431(4): 764-776, 2019 02 15.
Article En | MEDLINE | ID: mdl-30653991

Phosphopantothenoylcysteine (PPC) synthetase (PPCS) catalyzes nucleoside triphosphate-dependent condensation reaction between 4'-phosphopantothenate (PPA) and l-cysteine to form PPC in CoA biosynthesis. The catalytic mechanism of PPCS has not been resolved yet. Coenzyme A biosynthesis protein 2 (Cab2) possesses activity of PPCS in Saccharomyces cerevisiae. Our enzymatic assays suggest that Cab2 could utilize both ATP and CTP to activate PPA in vitro. The results of isothermal titration calorimetry indicate that PPA, CTP, and ATP could bind to Cab2 individually, with PPA having the highest binding affinity. To provide further insight into the catalytic mechanism of Cab2, we determined the crystal structures of Cab2 and its complex with PPA, the reaction intermediate 4'-phosphopantothenoyl-CMP, the final reaction product PPC, and the product analogue phosphopantothenoylcystine. Except for PPA, all other ligands were generated in situ and present in the active-site pocket of Cab2. Structures of Cab2 in complex with ligands provide insight into substrates binding and its catalytic mechanism. Analysis of structures indicates that the carboxyl of PPA-moiety of ligands and the γ-amino group of Asn97 possess different conformations in these complex structures. The cysteine/cystine/serine selectivity assays for Cab2 indicate that the amino group rather than the thiol group of l-cysteine attacks the carbonyl of 4'-phosphopantothenoyl-CMP to form PPC. Based on structural and biochemical data, the catalytic mechanism of Cab2 was proposed for the first time.


Peptide Synthases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Catalysis , Crystallography/methods , Protein Binding/physiology
16.
IUBMB Life ; 71(3): 330-339, 2019 03.
Article En | MEDLINE | ID: mdl-30501007

Catechol-O-methyltransferase (COMT1 ) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to various catechol substrates. COMTs play vital roles in physiological processes in animals, plants, and fungi, as well as bacteria, and have essential application values in industry. spCOMT is a probable COMT from Schizosaccharomyces pombe. It has an extraordinary intracellular distribution different from other homologs and would thus be predicted to perform a distinct physiological function. In this report, recombinant spCOMT was purified and kinetically characterized for the first time. The enzymology assays indicate that spCOMT is a metal-dependent enzyme and belongs to class I OMTs. In addition, the crystal structures of apo-spCOMT and SAM-complexed spCOMT were also presented, revealing that spCOMT possesses a conserved SAM-binding site and Mg2+ pocket, but a distinct substrate pocket was not present in homologs. The mutagenesis ITC analysis revealed the SAM recognition characteristics of spCOMT. Based on all of the above findings, we speculated about the putative substrates' characteristics and the substrate recognition mechanisms of spCOMT. This work will help in elucidating the physiological functions of spCOMT in S. pombe. © 2018 IUBMB Life, 71(3):330-339, 2019.


Catechol O-Methyltransferase/chemistry , Catechols/chemistry , Fungal Proteins/chemistry , S-Adenosylmethionine/chemistry , Schizosaccharomyces/chemistry , Amino Acid Sequence , Animals , Catalytic Domain , Catechol O-Methyltransferase/genetics , Catechol O-Methyltransferase/metabolism , Catechols/metabolism , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Kinetics , Mice , Models, Molecular , Mutation , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , S-Adenosylmethionine/metabolism , Schizosaccharomyces/enzymology , Schizosaccharomyces/genetics , Sequence Alignment , Structural Homology, Protein , Substrate Specificity
18.
Biochem Biophys Res Commun ; 503(4): 2943-2948, 2018 09 18.
Article En | MEDLINE | ID: mdl-30107915

By bearing a papain-like core structure and a cysteine-based catalytic triad, deamidase can convert glutamine to glutamic acid or asparagine to aspartic acid to modify the functions of host target proteins resulting in the blocking of eukaryotic host cell function. Legionella pneumophila effector Lpg2148 (MvcA) is a deamidase, a structural homolog of cycle inhibiting factor (Cif) effectors. Lpg2148 and Cif effectors are functionally diverse, with Lpg2148 only catalyzing ubiquitin but not NEDD8. However, a detailed understanding of substrate specificity is still missing. Here, we resolved the crystal structure of Lpg2148 at 2.5 Šresolution and obtained rigid-body modeling of Lpg2148 with C-terminus deleted ubiquitin (1-68) (ubΔc) complex using HADDOCK, which shows that the C-terminus of ubiquitin is flexible in recognition. We also conducted the truncated analysis to demonstrate that Leu71 of ubiquitin is necessary for its interaction with Lpg2148. Moreover, Val33 of Lpg2148 at the edge of a channel plays a vital role in the interaction and is limited by the length of the C-terminus of ubiquitin, which may help to explain the selectivity of ubiquitin over NEDD8. In summary, these results enrich our knowledge of substrate recognition of deamidase.


Amidohydrolases/metabolism , Ubiquitin/metabolism , HeLa Cells , Humans , Legionella pneumophila , NEDD8 Protein/metabolism , Substrate Specificity
19.
BMC Cancer ; 18(1): 248, 2018 03 02.
Article En | MEDLINE | ID: mdl-29499655

BACKGROUND: As hybrid RNAs, transcription-induced chimeras (TICs) may have tumor-promoting properties, and some specific chimeras have become important diagnostic markers and therapeutic targets for cancer. METHODS: We examined 23 paired laryngeal cancer (LC) tissues and adjacent normal mucous membrane tissue samples (ANMMTs). Three of these pairs were used for comparative transcriptomic analysis using high-throughput sequencing. Furthermore, we used real-time polymerase chain reaction (RT-PCR) for further validation in 20 samples. The Kaplan-Meier method and Cox regression model were used for the survival analysis. RESULTS: We identified 87 tumor-related TICs and found that COL7A1-UCN2 had the highest frequency in LC tissues (13/23; 56.5%), whereas none of the ANMMTs were positive (0/23; p < 0.0001). COL7A1-UCN2, generated via alternative splicing in LC tissue cancer cells, had disrupted coding regions, but it down-regulated the mRNA expression of COL7A1 and UCN2. Both COL7A1 and UCN2 were down-expressed in LC tissues as compared to their paired ANMMTs. The COL7A1:ß-actin ratio in COL7A1-UCN2-positive LC samples was significantly lower than that in COL7A1-UCN2-negative samples (p = 0.019). Likewise, the UCN2:ß-actin ratio was also decreased (p = 0.21). Furthermore, COL7A1-UCN2 positivity was significantly associated with the overall survival of LC patients (p = 0.032; HR, 13.2 [95%CI, 1.2-149.5]). CONCLUSION: LC cells were enriched in the recurrent chimera COL7A1-UCN2, which potentially affected cancer stem cell transition, promoted epithelial-mesenchymal transition in LC, and resulted in poorer prognoses.


Biomarkers, Tumor/genetics , Collagen Type VII/genetics , Corticotropin-Releasing Hormone/genetics , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing/methods , Laryngeal Neoplasms/genetics , Urocortins/genetics , Alternative Splicing , Case-Control Studies , Combined Modality Therapy , Computational Biology , Epithelial-Mesenchymal Transition , Female , Follow-Up Studies , Gene Expression Profiling , Humans , Laryngeal Neoplasms/pathology , Laryngeal Neoplasms/therapy , Male , Middle Aged , Prognosis , Survival Rate
20.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 1): 6-13, 2018 01 01.
Article En | MEDLINE | ID: mdl-29372902

Thiolases are vital enzymes which participate in both degradative and biosynthetic pathways. Biosynthetic thiolases catalyze carbon-carbon bond formation by a Claisen condensation reaction. The cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae, ERG10, catalyses carbon-carbon bond formation in the mevalonate pathway. The structure of a S. cerevisiae biosynthetic thiolase has not previously been reported. Here, crystal structures of apo ERG10 and its Cys91Ala variant were solved at resolutions of 2.2 and 1.95 Å, respectively. The structure determined shows that ERG10 shares the characteristic thiolase superfamily fold, with a similar active-site architecture to those of type II thiolases and a similar binding pocket, apart from Ala159 at the entrance to the pantetheine-binding cavity, which appears to be a determinant of the poor binding ability of the substrate. Moreover, comparative binding-pocket analysis of molecule B in the asymmetric unit of the apo structure with that of the CoA-bound complex of human mitochondrial acetoacetyl-CoA thiolase indicates the canonical binding mode of CoA. Furthermore, the steric hindrance revealed in a structural comparison of molecule A with the CoA-bound form raise the possibility of conformational changes that are associated with substrate binding.


Acetyl-CoA C-Acetyltransferase/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Acetyl-CoA C-Acetyltransferase/genetics , Amino Acid Sequence , Amino Acid Substitution , Catalytic Domain , Crystallography, X-Ray , Cytoplasm/enzymology , Genes, Fungal , Humans , Models, Molecular , Mutagenesis, Site-Directed , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Species Specificity , Static Electricity , Structural Homology, Protein
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