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1.
Proc Natl Acad Sci U S A ; 115(39): E9105-E9114, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30209215

RESUMO

Integrin α/ß heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of ß subunit induce the α/ß separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The ß3 integrins, including αIIbß3 and αVß3, are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the ß3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti-HPA-1a alloantibodies can distinguish the αIIbß3 from αVß3 and affect their functions with unknown mechanisms. Here we designed a single-chain ß3 subunit that mimics a separation of α/ß heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain ß3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the ß3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain ß3 integrin, we identified the conformation-dependent property of anti-HPA-1a alloantibodies, which enables them to differently recognize the ß3 in the bent state vs. the extended state and in the complex with αIIb vs. αV This study provides deeper understandings of integrin conformational activation on the cell surface.


Assuntos
Glucuronidase/química , Integrina beta3/química , Isoanticorpos/química , Especificidade de Anticorpos , Cristalografia por Raios X , Glucuronidase/metabolismo , Células HEK293 , Humanos , Integrina alfaVbeta3/química , Integrina alfaVbeta3/metabolismo , Integrina beta3/metabolismo , Isoanticorpos/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Domínios Proteicos , Dobramento de Proteína
2.
Blood ; 132(9): 962-972, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30018079

RESUMO

Cells use adhesion receptor integrins to communicate with their surroundings. Integrin activation and cellular signaling are coupled with change from bent to extended conformation. ß3 integrins, including αIIbß3, which is essential for the function of platelets in hemostasis and thrombosis, and αVß3, which plays multiple roles in diverse cell types, have been prototypes in understanding integrin structure and function. Despite extensive structural studies, a high-resolution integrin structure in an extended conformation remains to be determined. The human ß3 Leu33Pro polymorphism, located at the PSI domain, defines human platelet-specific alloantigens 1a and 1b (HPA-1a/b), immune response to which is a cause of posttransfusion purpura and fetal/neonatal alloimmune thrombocytopenia. Leu33Pro substitution has also been suggested to be a risk factor for thrombosis. Here we report the crystal structure of the ß3 headpiece in either Leu33 or Pro33 form, both of which reveal intermediate and fully extended conformations coexisting in 1 crystal. These were used to build high-resolution structures of full-length ß3 integrin in the intermediate and fully extended states, agreeing well with the corresponding conformations observed by electron microscopy. Our structures reveal how ß3 integrin becomes extended at its ß-knee region and how the flexibility of ß-leg domains is determined. In addition, our structures reveal conformational changes of the PSI and I-EGF1 domains upon ß3 extension, which may affect the binding of conformation-dependent anti-HPA-1a alloantibodies. Our structural and functional data show that Leu33Pro substitution does not directly alter the conformation or ligand binding of ß3 integrin.


Assuntos
Integrina beta3/química , Polimorfismo Genético , Substituição de Aminoácidos , Células HEK293 , Humanos , Integrina beta3/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
3.
Blood ; 127(8): 1052-61, 2016 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-26702061

RESUMO

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily (IgSF) that is present on the surface of circulating platelets and leukocytes, and highly expressed at the junctions of confluent endothelial cell monolayers. PECAM-1-mediated homophilic interactions, known to be mediated by its 2 amino-terminal immunoglobulin homology domains, are essential for concentrating PECAM-1 at endothelial cell intercellular junctions, where it functions to facilitate diapedesis, maintain vascular integrity, and transmit survival signals into the cell. Given the importance of PECAM-1-mediated homophilic interactions in mediating each of these cell physiological events, and to reveal the nature and orientation of the PECAM-1-PECAM-1 homophilic-binding interface, we undertook studies aimed at determining the crystal structure of the PECAM-1 homophilic-binding domain, which is composed of amino-terminal immunoglobulin homology domains 1 and 2 (IgD1 and IgD2). The crystal structure revealed that both IgD1 and IgD2 exhibit a classical IgSF fold, having a ß-sandwich topology formed by 2 sheets of antiparallel ß strands stabilized by the hallmark disulfide bond between the B and F strands. Interestingly, despite previous assignment to the C2 class of immunoglobulin-like domains, the structure of IgD1 reveals that it actually belongs to the I2 set of IgSF folds. Both IgD1 and IgD2 participate importantly in the formation of the trans homophilic-binding interface, with a total buried interface area of >2300 Å(2). These and other unique structural features of PECAM-1 allow for the development of an atomic-level model of the interactions that PECAM-1 forms during assembly of endothelial cell intercellular junctions.


Assuntos
Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Modelos Moleculares , Molécula-1 de Adesão Celular Endotelial a Plaquetas/química , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular/fisiologia , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Especificidade da Espécie , Transfecção
4.
Biochemistry ; 52(12): 2148-56, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23448527

RESUMO

CrataBL, a protein isolated from Crataeva tapia bark, which is both a serine protease inhibitor and a lectin, has been previously shown to exhibit a number of interesting biological properties, including anti-inflammatory, analgesic, antitumor, and insecticidal activities. Using a glycan array, we have now shown that only sulfated carbohydrates are effectively bound by CrataBL. Because this protein was recently shown to delay clot formation by impairing the intrinsic pathway of the coagulation cascade, we considered that its natural ligand might be heparin. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins, including thrombin and antithrombin III, which have a critical, essential pharmacological role in regulating blood coagulation. We have thus employed surface plasmon resonance to improve our understanding of the binding interaction between the heparin polysaccharide and CrataBL. Kinetic analysis shows that CrataBL displays strong heparin binding affinity (KD = 49 nM). Competition studies using different size heparin-derived oligosaccharides showed that the binding of CrataBL to heparin is chain length-dependent. Full chain heparin with 40 saccharides or large oligosaccharides, having 16-18 saccharide residues, show strong binding affinity for CrataBL. Heparin-derived disaccharides through tetradecasaccharides show considerably lower binding affinity. Other highly sulfated GAGs, including chondroitin sulfate E and dermatan 4,6-disulfate, showed CrataBL binding affinity comparable to that of heparin. Less highly sulfated GAGs, heparan sulfate, chondroitin sulfate A and C, and dermatan sulfate displayed modest binding affinity as did chondroitin sulfate D. Studies using chemically modified heparin show that N-sulfo and 6-O-sulfo groups on heparin are essential for CrataBL-heparin interaction.


Assuntos
Capparaceae/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Capparaceae/genética , Glicosaminoglicanos/química , Heparina/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/genética , Ligação Proteica , Conformação Proteica , Ressonância de Plasmônio de Superfície
5.
J Struct Biol ; 177(3): 638-45, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22366278

RESUMO

The ribonuclease H (RNase H) domain of retroviral reverse transcriptase (RT) plays a critical role in the life cycle by degrading the RNA strands of DNA/RNA hybrids. In addition, RNase H activity is required to precisely remove the RNA primers from nascent (-) and (+) strand DNA. We report here three crystal structures of the RNase H domain of xenotropic murine leukemia virus-related virus (XMRV) RT, namely (i) the previously identified construct from which helix C was deleted, (ii) the intact domain, and (iii) the intact domain complexed with an active site α-hydroxytropolone inhibitor. Enzymatic assays showed that the intact RNase H domain retained catalytic activity, whereas the variant lacking helix C was only marginally active, corroborating the importance of this helix for enzymatic activity. Modeling of the enzyme-substrate complex elucidated the essential role of helix C in binding a DNA/RNA hybrid and its likely mode of recognition. The crystal structure of the RNase H domain complexed with ß-thujaplicinol clearly showed that coordination by two divalent cations mediates recognition of the inhibitor.


Assuntos
Cristalografia por Raios X/métodos , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/química , Ribonuclease H/metabolismo , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/enzimologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-21543876

RESUMO

The RadA intein from the hyperthermophilic archaebacterium Pyrococcus horikoshii was cloned, expressed and purified for subsequent structure determination. The protein crystallized rapidly in several conditions. The best crystals, which diffracted to 1.75 Å resolution, were harvested from drops consisting of 0.1 M HEPES pH 7.5, 3.0 M NaCl and were cryoprotected with Paratone-N before flash-cooling. The collected data were processed in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 58.1, b = 67.4, c = 82.9 Å. Molecular replacement with Rosetta using energy- and density-guided structure optimization provided the initial solution, which is currently under refinement.


Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Pyrococcus horikoshii/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Expressão Gênica , Dados de Sequência Molecular
7.
3 Biotech ; 10(4): 166, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32206500

RESUMO

The natural estrogen 17ß-estradiol (17ß-E2) is a major endocrine disruptor. Accordingly, due to their frequent presence in global surface waters, prolonged exposure to estrogen-contaminated water may disrupt sexual development in animals. It has adverse effects on wildlife and humans. To date, the most effective strategy for estrogen removal from the environment is biodegradation using microorganisms. To this end, we isolated a strain of Lysinibacillus sphaericus, namely DH-B01, from a contraceptive factory in Beijing. The experimental results revealed that the bacterium has a high capacity to degrade estrogen, with a 17ß-E2 degradation rate of about 97%, and produces the secondary metabolite estrone. In addition, a series of genes involved in steroid metabolism and stress response in L. sphaericus sp. DH-B01 were predicted, and several key genes with high similarity to those of other strains were subjected to sequence alignment to find their conserved regions. This is the first study of the ability of L. sphaericus strains to degrade estrogens and the degradation mechanism involved. This work advances the genomic study of estrogen-degrading strains and the study of bacterial estrogen degradation mechanisms. In this paper, a novel bacterial strain capable of degrading 17ß-E2 was studied. L. sphaericus sp. DH-B01 can effectively degrade 17ß-E2. During the degradation process, 17ß-E2 can be gradually metabolized to a substance without estrogen activity. By analyzing the enzymatic reactions in the metabolic process, we found genes with high similarity to reported 17ß-HSD. L. sphaericus sp. DH-B01 was found to degrade 17ß-E2. There are many types of bacteria that are currently being studied for the degradation of estrogen, but L. sphaericus sp. DH-B01 is the only strain of L. sphaericus that has been shown to degrade estrogen. This work advances the genomic study of estrogen-degrading bacterial strains and the study of bacterial estrogen degradation mechanisms. Additionally, it explores the correlation between different L. sphaericus strains. The differences play an important role and further enrich the functionality and diversity of L. sphaericus strains. In subsequent studies, the specificity of L. sphaericus sp. DH-B01 can be applied to different environments for future environmental restoration.

8.
Zhong Yao Cai ; 30(2): 155-7, 2007 Feb.
Artigo em Zh | MEDLINE | ID: mdl-17571759

RESUMO

A kind of glycoprotein ( CF- I ) was extracted from Cimicifuga foetida and purified by DEAE-Cellulose ( DEAE-52) and Sepharose CL-4B. It was identified to be homogeneous glycoprotein complex by electrophoresis and fast protein liquid chromatography (FPLC). alpha-glucosidic bond was detrermined by IR. Typical absorption of polysaccharide was shown in its IR spectrum. It had no typical absorption of nucleic acid or pigment by UV scanning. Glucose, galactose, mannose and arabinose were identified in CF- I with the molar ratio of 11. 94: 2. 18: 1. 38: 1 by GC. Its average MW was estimated to be 5. 8 x 10' by gel filtration. The content of total saccharide, protein and acid polysaccharide was 78% , 14. 4% and 23% respectively. It might be composed of 1-->4 and 1-->6 linked glucopyranose by periodate oxidation and Smith degration.


Assuntos
Cimicifuga/química , Glicoproteínas/isolamento & purificação , Plantas Medicinais/química , Cromatografia Líquida de Alta Pressão , Galactose/análise , Galactose/química , Glucose/análise , Glucose/química , Glicoproteínas/análise , Glicoproteínas/química , Temperatura Alta , Estrutura Molecular , Peso Molecular , Rizoma/química , Espectrofotometria Ultravioleta , Ácidos Urônicos/análise , Ácidos Urônicos/química
9.
J Mol Biol ; 429(24): 3942-3956, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-29055778

RESUMO

Inteins are mobile genetic elements that are spliced out of proteins after translation. Some inteins contain a homing endonuclease (HEN) responsible for their propagation. Hedgehog/INTein (HINT) domains catalyzing protein splicing and their nested HEN domains are thought to be functionally independent because of the existence of functional mini-inteins without HEN domains. Despite the lack of obvious mutualism between HEN and HINT domains, HEN domains are persistently found at one specific site in inteins, indicating their potential functional role in protein splicing. Here we report crystal structures of inactive and active mini-inteins derived from inteins residing in the transcription factor IIB of Methanococcus jannaschii and Methanocaldococcus vulcanius, revealing a novel modified HINT fold that might provide new insights into the mutualism between the HEN and HINT domains. We propose an evolutionary model of inteins and a functional role of HEN domains in inteins.


Assuntos
Endonucleases/química , Inteínas , Mathanococcus/enzimologia , Fator de Transcrição TFIIB/química , Sequência de Aminoácidos , Cristalografia por Raios X , Endonucleases/genética , Endonucleases/metabolismo , Mathanococcus/genética , Modelos Moleculares , Conformação Proteica , Processamento de Proteína , Homologia de Sequência , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo
10.
Artigo em Inglês | MEDLINE | ID: mdl-16682780

RESUMO

Galectins are a family of animal lectins which share similar carbohydrate-recognition domains (CRDs) and an affinity for beta-galactosides. A novel human galectin-related protein named GRP (galectin-related protein; previously known as HSPC159) comprises only one conserved CRD with 38 additional N-terminal residues. The C-terminal fragment of human GRP (GRP-C; residues 38-172) containing the CRD has been expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 2% PEG 400 and 2M ammonium sulfate in 100 mM Tris-HCl buffer pH 7.5. Diffraction data were collected to a resolution limit of 2.0 angstroms at beamline 3W1A of Beijing Synchrotron Radiation Facility at 100 K. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 123.07, b = 96.67, c = 61.56 angstroms, beta = 118.72 degrees. The estimated Matthews coefficient was 2.6 angstroms3 Da(-1), corresponding to 51.8% solvent content.


Assuntos
Galectinas/biossíntese , Galectinas/química , Lectinas/biossíntese , Lectinas/química , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Humanos , Estrutura Terciária de Proteína
11.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 8): 1055-62, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26249699

RESUMO

A serine protease inhibitor from Bauhinia bauhinioides (BbKI) belongs to the Kunitz family of plant inhibitors, which are common in plant seeds. BbKI does not contain any disulfides, unlike most other members of this family. It is a potent inhibitor of plasma kallikrein, in addition to other serine proteases, and thus exhibits antithrombotic activity. A high-resolution crystal structure of recombinantly expressed BbKI was determined (at 1.4 Šresolution) and was compared with the structures of other members of the family. Modeling of a complex of BbKI with plasma kallikrein indicates that changes in the local structure of the reactive loop that includes the specificity-determining Arg64 are necessary in order to explain the tight binding. An R64A mutant of BbKI was found to be a weaker inhibitor of plasma kallikrein, but was much more potent against plasmin, suggesting that this mutant may be useful for preventing the breakup of fibrin and maintaining clot stability, thus preventing excessive bleeding.


Assuntos
Bauhinia/química , Fibrinolisina/antagonistas & inibidores , Fibrinolíticos/química , Proteínas de Plantas/química , Calicreína Plasmática/antagonistas & inibidores , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Fibrinolisina/química , Fibrinolíticos/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/genética , Calicreína Plasmática/química , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sementes/química , Homologia Estrutural de Proteína
12.
Mol Biosyst ; 10(5): 1023-34, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24574026

RESUMO

Protein splicing is an autocatalytic process involving self-excision of an internal protein domain, the intein, and concomitant ligation of the two flanking sequences, the exteins, with a peptide bond. Protein splicing can also take place in trans by naturally split inteins or artificially split inteins, ligating the exteins on two different polypeptide chains into one polypeptide chain. Protein trans-splicing could work in foreign contexts by replacing the native extein sequences with other protein sequences. Protein ligation using protein trans-splicing increasingly becomes a useful tool for biotechnological applications such as semi-synthesis of proteins, segmental isotopic labeling, and in vivo protein engineering. However, only a few split inteins have been successfully applied for protein ligation. Naturally split inteins have been widely used, but they are cross-reactive to each other, limiting their applications to multiple-fragment ligation. Based on the three-dimensional structures including two newly determined intein structures, we derived 21 new split inteins from four highly efficient cis-splicing inteins, in order to develop novel split inteins suitable for protein ligation. We systematically compared trans-splicing of 24 split inteins and tested the cross-activities among them to identify orthogonal split intein fragments that could be used in chemical biology and biotechnological applications.


Assuntos
Inteínas , Engenharia de Proteínas/métodos , Processamento de Proteína , Cristalografia por Raios X , Cinética , Modelos Biológicos , Modelos Moleculares , Terminologia como Assunto , Trans-Splicing
13.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 7): 942-5, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25005094

RESUMO

Histamine dehydrogenase (HADH) catalyzes the oxidative deamination of histamine, resulting in the production of imidazole acetaldehyde and an ammonium ion. The enzyme isolated from the newly identified halophilic archaeon Natrinema gari BCC 24369 is significantly different from the previously described protein from Nocardioides simplex. This newly identified HADH comprises three subunits with molecular weights of 49.0, 24.7 and 23.9 kDa, respectively, and is optimally active under high-salt conditions (3.5-5 M NaCl). As a step in the exploration of the unique properties of the protein, the HADH heterotrimer was purified and crystallized. Crystals were obtained using the sitting-drop vapor-diffusion method from a solution composed of 0.2 M calcium chloride dihydrate, 0.1 M HEPES pH 7.5, 28% PEG 400. Diffraction data were collected at -173°C to a resolution limit of 2.4 Šon the Southeast Regional Collaborative Access Team (SER-CAT) beamline 22-ID at the Advanced Photon Source, Argonne National Laboratory. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=211.9, b=58.6, c=135.4 Å, ß=103.0°. The estimated Matthews coefficient is 3.21 Å3 Da(-1), corresponding to 61.7% solvent content.


Assuntos
Proteínas Arqueais/química , Halobacteriaceae/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Subunidades Proteicas/química , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/metabolismo , Cristalização , Cristalografia por Raios X , Expressão Gênica , Halobacteriaceae/enzimologia , Halobacteriaceae/genética , Peso Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo
16.
PLoS One ; 8(4): e62252, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23626794

RESUMO

A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1' pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.


Assuntos
Fabaceae/química , Inibidores da Tripsina/química , Tripsina/química , Sequência de Aminoácidos , Animais , Domínio Catalítico , Bovinos , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Serina Proteases/química , Serina Proteases/metabolismo , Tripsina/metabolismo , Inibidores da Tripsina/metabolismo
17.
PLoS One ; 8(6): e64426, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23823708

RESUMO

A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (Kiapp = 43 µM) and was more potent against Factor Xa (Kiapp = 8.6 µM), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. The high-resolution crystal structures of two different crystal forms of isoform II verified the ß-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (∼645 Å(2)). The structure differs from those of the most closely related proteins by the lack of the N-terminal ß-hairpin. In experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 µM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). The apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells.


Assuntos
Capparaceae/química , Lectinas de Plantas/farmacologia , Sequência de Aminoácidos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Dimerização , Ensaios de Seleção de Medicamentos Antitumorais , Glicosilação , Humanos , Masculino , Estrutura Molecular , Lectinas de Plantas/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
J Mol Biol ; 421(1): 85-99, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22560994

RESUMO

In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the extein that follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein from Pyrococcus horikoshii (PhoRadA). The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of a less robust splicing variant by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offer a promising way of improving their properties as better tools for protein engineering.


Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Inteínas , Pyrococcus horikoshii/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Exteínas/genética , Inteínas/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Engenharia de Proteínas/métodos , Processamento de Proteína
19.
Nat Struct Mol Biol ; 18(2): 227-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21258323

RESUMO

Using energy and density guided Rosetta refinement to improve molecular replacement, we determined the crystal structure of the protease encoded by xenotropic murine leukemia virus-related virus (XMRV). Despite overall similarity of XMRV protease to other retropepsins, the topology of its dimer interface more closely resembles those of the monomeric, pepsin-like enzymes. Thus, XMRV protease may represent a distinct branch of the aspartic protease family.


Assuntos
Peptídeo Hidrolases/química , Proteínas Virais/química , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Dados de Sequência Molecular , Multimerização Proteica , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/química
20.
PLoS One ; 6(12): e27751, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22145019

RESUMO

BACKGROUND: We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins. METHODOLOGY/RESULTS: Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate. CONCLUSION: Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs. a variety of interactions that might occur during formation of functional IN complexes in solution.


Assuntos
Vírus do Sarcoma Aviário/enzimologia , Reagentes de Ligações Cruzadas/farmacologia , DNA Viral/química , DNA Viral/metabolismo , Integrases/química , Integrases/metabolismo , Sequência de Aminoácidos , Vírus do Sarcoma Aviário/genética , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA Viral/genética , Integrases/genética , Luz , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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