RESUMO
Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.
Assuntos
Araceae/enzimologia , Modelos Moleculares , Peroxidase/química , Conformação Proteica , Sequência de Aminoácidos , Sequência de Bases , Cristalização , Primers do DNA/genética , DNA Complementar/genética , Glicosilação , Cinética , Dados de Sequência Molecular , Peroxidase/genética , Peroxidase/metabolismo , Análise de Sequência de DNA , Espectrometria de Massas em TandemRESUMO
Interleukin-22 (IL-22) plays an important role in the regulation of immune and inflammatory responses in mammals. The IL-22 binding protein (IL-22BP), a soluble receptor that specifically binds IL-22, prevents the IL-22/interleukin-22 receptor 1 (IL-22R1)/interleukin-10 receptor 2 (IL-10R2) complex assembly and blocks IL-22 biological activity. Here we present the crystal structure of the IL-22/IL-22BP complex at 2.75 A resolution. The structure reveals IL-22BP residues critical for IL-22 binding, which were confirmed by site-directed mutagenesis and functional studies. Comparison of IL-22/IL-22BP and IL-22/IL-22R1 crystal structures shows that both receptors display an overlapping IL-22 binding surface, which is consistent with the inhibitory role played by IL-22 binding protein.
Assuntos
Interleucinas/química , Receptores de Interleucina/química , Sítios de Ligação/fisiologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Subunidade beta de Receptor de Interleucina-10/química , Subunidade beta de Receptor de Interleucina-10/genética , Subunidade beta de Receptor de Interleucina-10/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Interleucina 22RESUMO
Interleukin-22 (IL-22) is a pleiotropic cytokine that is involved in inflammatory responses. Human IL-22 was incubated with its soluble decoy receptor IL-22BP (IL-22 binding protein) and the IL-22-IL-22BP complex was crystallized in hanging drops using the vapour-diffusion method. Suitable crystals were obtained from polyethylene glycol solutions and diffraction data were collected to 2.75 A resolution. The crystal belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 67.9, c = 172.5 A, and contained two IL-22-IL-22BP complexes per asymmetric unit.
Assuntos
Interleucinas/química , Interleucinas/metabolismo , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Difração de Raios X , Cristalização , Humanos , Modelos Moleculares , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Difração de Raios X/métodos , Interleucina 22RESUMO
Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2.
Assuntos
Interleucinas/química , Receptores de Interleucina/química , Sequência de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Humanos , Interleucinas/genética , Dados de Sequência Molecular , Mutação , Conformação Proteica , Receptores de Interleucina/genética , Receptores de Interleucina-10/química , Transdução de Sinais , Interleucina 22RESUMO
Royal palm tree peroxidase (RPTP), which was isolated from Roystonea regia leaves, has an unusually high stability that makes it a promising candidate for diverse applications in industry and analytical chemistry [Caramyshev et al. (2005), Biomacromolecules, 6, 1360-1366]. Here, the purification and crystallization of this plant peroxidase and its X-ray diffraction data collection are described. RPTP crystals were obtained by the hanging-drop vapour-diffusion method and diffraction data were collected to a resolution of 2.8 A. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 116.83, c = 92.24 A, and contain one protein molecule per asymmetric unit. The V(M) value and solvent content are 4.07 A3 Da(-1) and 69.8%, respectively.
Assuntos
Arecaceae/enzimologia , Peroxidases/química , Cristalização , Peso Molecular , Peroxidases/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Árvores/enzimologia , Difração de Raios XRESUMO
The crystal structure of dimeric Lys49-phospholipase A2 myotoxin-II from Bothrops moojeni (MjTX-II) co-crystallized with stearic acid (C(18)H(36)O(2)) has been determined at a resolution of 1.8 A. The electron density maps permitted the unambiguous inclusion of six stearic acid molecules in the refinement. Two stearic acid molecules could be located in the substrate-binding cleft of each monomer in positions, which favor the interaction of their carboxyl groups with active site residues. The way of binding of stearic acids to this Lys49-PLA(2)s is analogous to phospholipids and transition state analogues to catalytically active PLA(2)s. Two additional stearic acid molecules were located at the dimer interface region, defining a hitherto unidentified acyl-binding site on the protein surface. The strictly conserved Lys122 for Lys49-PLA(2)s may play a fundamental role for stabilization of legend-protein complex. The comparison of MjTX-II/satiric acid complex with other Lys-PLA(2)s structures whose putative fatty acids were located at their active site is also analysed. Molecular details of the stearic acid/protein interactions provide insights to binding in group I/II PLA(2)s, and to the possible interactions of Lys49-PLA(2)s with target membranes.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Fosfolipases A/química , Ácidos Esteáricos/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Fosfolipases A2 do Grupo II , Estrutura Terciária de ProteínaRESUMO
A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bb chain of fibrinogen while the Aa chain and g chain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bb chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mm x 0.2 mm x 0.2 mm) and used for X-ray diffraction experiments.
Assuntos
Venenos de Crotalídeos/enzimologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Venenos de Víboras/genética , Venenos de Víboras/metabolismo , Animais , Coagulação Sanguínea/efeitos dos fármacos , Bothrops , Cromatografia de Afinidade , Cristalização , Eletroforese em Gel de Poliacrilamida , Fibrinogênio/metabolismoRESUMO
Myotoxin II, a Lys49 catalytically inactive phospholipase A(2) homologue from Atropoides nummifer venom, was purified, characterized and crystallized. The crystals belongs to the tetragonal system, space group P4(3)2(1)2, with unit cell parameters (a=b=68.66 and c=63.87 angstroms). Diffraction data were collected to a resolution of 2.32 angstroms. The crystal structure is currently being determined using molecular replacement techniques.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Lisina/química , Fosfolipases A/análise , Fosfolipases A/química , Animais , Cristalografia por Raios X , Fosfolipases A2 do Grupo II , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Proteínas de Répteis , Difração de Raios XRESUMO
BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.
Assuntos
Bothrops , Venenos de Crotalídeos/enzimologia , Metaloendopeptidases/química , Sequência de Aminoácidos , Animais , Cálcio/química , Domínio Catalítico/fisiologia , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Cristalografia por Raios X , Hemorragia/etiologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Zinco/químicaRESUMO
A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoelectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIHU/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degrees C and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 degrees C. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4A.