RESUMO
Dipeptidyl peptidase IV (DPP IV, DPP4, or DAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position. The substrate recognition mechanism has been fully elucidated for mammalian DPP IV by crystal structure analyses but not for bacterial orthologues. Here, we report the crystal structures of a bacterial DPP IV (PmDAP IV) in its free form and in complexes with two kinds of dipeptides as well as with a non-peptidyl inhibitor at 1.90 to 2.47 Å resolution. Acyl-enzyme intermediates were observed for the dipeptide complexes of PmDAP IV, whereas tetrahedral intermediates were reported for the oligopeptide complexes of mammalian DPP IVs. This variation reflects the different structural environments of the active site Arg residues, which are involved in the recognition of a substrate carbonyl group, of mammalian and bacterial enzymes. A phylogenetic analysis revealed that PmDAP IV is a closer relative of dipeptidyl peptidases 8 and 9 (DPP8 and DPP9, DPP IV-family enzymes) than DPP IV. These results provide new insights into the substrate recognition mechanism of bacterial DAP IVs and may assist in the development of selective inhibitors for DAP IVs from pathogenic asaccharolytic bacteria, which utilise proteins or peptides as an energy source.
Assuntos
Dipeptídeos/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/metabolismo , Xanthomonadaceae/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Dipeptidil Peptidase 4/genética , Humanos , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Especificidade por SubstratoRESUMO
Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2' ]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90â Å resolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84â Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.
Assuntos
Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/isolamento & purificação , Periplasma/enzimologia , Xanthomonadaceae/enzimologia , Cristalização , Cristalografia por Raios X , Dipeptidil Peptidase 4/metabolismo , Cinética , Conformação Proteica , Xanthomonadaceae/classificaçãoRESUMO
S-adenosyl-L-homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L-homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues-3'-keto-aristeromycin (3KA) and noraristeromycin (NRN)-at resolutions of 1.55, 1.55, and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3'-keto-4',5'-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer &Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme.
Assuntos
Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Hidrólise , Camundongos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína , S-Adenosil-Homocisteína/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of this malaria parasite resistant to conventional drug therapy has stimulated the search for antimalarial compounds with novel modes of action. Here the structure-function relationship studies for two Plasmodium proteins are presented. One example is the structural studies for S-adenosyl-L-homocysteine hydrolase from Plasmodium falciparum (PfSAHH) and the other example is those for 1-deoxy-D-xylulose reductoisomerase from Plasmodium falciparum (PfDXR). In the former study, the clue for design of species specific PfSAHH inhibitors was obtained by the structural comparison of the active site of PfSAHH with that of human SAHH (HsSAHH). Our study revealed that the inhibitor selectivity depends on the difference of only one amino acid residue in the active site; Cys59 in PfSAHH vs. Thr60 in HsSAHH. In the latter study, the inhibition of PfDXR enzyme by fosmidomycin has proved to be efficient in the treatment of uncomplicated malaria in recent clinical trials conducted in Gabon and Thailand. Our crystal structure analyses of PfDXR/inhibitor complexes revealed the molecular basis of fosmidomycin's action in P. falciparum. We expect that the structure-function relationship studies on Plasmodium proteins are useful for developing the more effective antimalarial compounds.
Assuntos
Adenosil-Homocisteinase/antagonistas & inibidores , Aldose-Cetose Isomerases/antagonistas & inibidores , Antimaláricos/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Plasmodium falciparum/enzimologia , Plasmodium falciparum/genética , Animais , Antimaláricos/uso terapêutico , Domínio Catalítico , Ensaios Clínicos como Assunto , Cristalização , Cristalografia , Cisteína , Fosfomicina/análogos & derivados , Fosfomicina/uso terapêutico , Humanos , Conformação Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , TreoninaRESUMO
Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0â Å resolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9â Å, ß = 122.6°.
Assuntos
Complexos Multienzimáticos/química , Fosfodiesterase I/química , Pirofosfatases/química , Cristalização , Cristalografia por Raios X , Humanos , Diester Fosfórico HidrolasesRESUMO
S-adenosyl-L-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 A resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 A. Structural analysis by molecular replacement is in progress.
Assuntos
Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/genética , Adenosil-Homocisteinase/isolamento & purificação , Animais , Cristalografia por Raios X , Expressão Gênica , CamundongosRESUMO
Phosphoglucose isomerase (PGI) is a key enzyme in glycolysis and glycogenesis that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). For crystallographic studies, PGI from the human malaria parasite Plasmodium falciparum (PfPGI) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.5 A resolution were collected from an orthorhombic crystal form belonging to space group P2(1)2(1)2(1) with unit-cell parameters a = 103.3, b = 104.1, c = 114.6 A. Structural analysis by molecular replacement is in progress.
Assuntos
Glucose-6-Fosfato Isomerase/química , Plasmodium falciparum/enzimologia , Cristalização , Cristalografia por Raios X , Glucose-6-Fosfato Isomerase/isolamento & purificaçãoAssuntos
Exopeptidases/química , Exopeptidases/fisiologia , Peptídeos/metabolismo , Aminoácidos/metabolismo , Animais , Antígenos CD13/química , Antígenos CD13/genética , Antígenos CD13/fisiologia , Cristalografia por Raios X , Humanos , Mutação , Prolil Oligopeptidases , Serina Endopeptidases/química , Serina Endopeptidases/fisiologia , Especificidade por SubstratoRESUMO
Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and alpha-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C(19)/C(21)-steroids into 3beta-hydroxysteroids. The stereospecific conversion to 3beta-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor alpha ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3beta-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.
Assuntos
Hidroxiesteroide Desidrogenases/química , Hidroxiesteroide Desidrogenases/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Temperatura Baixa , Ativação Enzimática , Estabilidade Enzimática , Humanos , Distribuição TecidualRESUMO
Autotaxin (ATX) is an approximately 125kDa transmembrane protein known as a tumor progression factor based on its lysophospholipase D (lysoPLD) activity. There are many reports of the biological and biochemical properties of ATX, but crystallographic or structural studies have not been reported because a large-scale production process using prokaryotic cells has not been established. Here we report a bulk purification process and soluble expression of the recombinant human ATX (rhATX S48) from prokaryotic cells. The extracellular domain of human ATX cDNA was cloned into a pET101/D-TOPO vector and transformed to an Escherichia coliBL21 strain which was co-transformed with a pTF16 chaperone plasmid. The rhATX S48 was purified with chaperone and it was removed by Mg(2+)-ATP treatment. The final yield of purified rhATX S48 was approximately 3.5mg/l culture of recombinant strain. The rhATX S48 shows lysoPLD enzymatic activity and effectively stimulates the growth and motile activity of the human tumor cells as well as native ATX. This is a first report for scalable purification of the ATX molecule and the rhATX S48 should be a good tool for immunization of anti-ATX or crystallographic analysis of ATX.
Assuntos
Complexos Multienzimáticos/isolamento & purificação , Fosfodiesterase I/isolamento & purificação , Pirofosfatases/isolamento & purificação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cromatografia em Gel , Clonagem Molecular , Escherichia coli/metabolismo , Fibrossarcoma , Humanos , Complexos Multienzimáticos/metabolismo , Fosfodiesterase I/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Structural biology teaches us about the fundamental nature of biological molecules. Drug design is the most immediate medical application of structural biology. Therefore our studies have been focused on structural and functional studies of human disease-related proteins and proteins essential for the growth and development of pathogenic organisms. The present paper describes 1) structural biological studies of human autocrine motility factor, 2) structural biological studies of human ribonuclease L, and 3) structural biological studies of Plasmodium falciparum S-adenosyl- L-homocysteine hydrolase.
Assuntos
Adenosil-Homocisteinase/química , Adenosil-Homocisteinase/fisiologia , Desenho de Fármacos , Endorribonucleases/química , Endorribonucleases/fisiologia , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/fisiologia , Plasmodium falciparum/enzimologia , Adenosil-Homocisteinase/antagonistas & inibidores , Animais , Cristalografia por Raios X , Endorribonucleases/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Humanos , Conformação ProteicaRESUMO
The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.
Assuntos
Glucose-6-Fosfato Isomerase/metabolismo , Glucose/metabolismo , Receptores de Citocinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Glucose-6-Fosfato Isomerase/química , Glucose-6-Fosfato Isomerase/genética , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Receptores do Fator Autócrino de Motilidade , Receptores de Citocinas/química , Receptores de Citocinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Autocrine motility factor (AMF), a tumor-secreted cytokine, stimulates cell migration in vitro and metastasis in vivo. AMF is identical to the extracellular cytokines neuroleukin and maturation factor and, interestingly, to the intracellular enzyme phosphoglucose isomerase. The cytokine activity of AMF is inhibited by carbohydrate phosphate compounds as they compete for AMF binding with the carbohydrate moiety of the AMF receptor (AMFR), which is a glycosylated seven transmembrane helix protein. Here, we report the first comprehensive high-resolution crystal structure analyses of the inhibitor-free form and the eight types of inhibitor (phosphate, erythrose 4-phosphate (E4P), arabinose 5-phosphate (A5P), sorbitol 6-phosphate (S6P), 6-phosphogluconic acid (6PGA), fructose 6-phosphate (F6P), glucose 6-phosphate (G6P), or mannose 6-phosphate (M6P)) complexes of mouse AMF (mAMF). We assayed the inhibitory activities of these inhibitors against the cytokine activity of mAMF. The inhibitory activities of the six-carbon sugars (G6P, F6P, M6P, and 6PGA) were found to be significantly higher than those of the four or five-carbon sugars (E4P or A5P). The inhibitory activities clearly depend on the length of the inhibitor molecules. A structural comparison revealed that a water-mediated hydrogen bond between one end of the inhibitor and a rigid portion of the protein surface in the shorter-chain inhibitor (E4P) complex is replaced by a direct hydrogen bond in the longer-chain inhibitor (6PGA) complex. Thus, to obtain a new compound with higher inhibitory activities against AMF, water molecules at the inhibitor binding site of AMF should be replaced by a functional group of inhibitors in order to introduce direct interactions with the protein surface. The present structure-activity relationship studies will be valuable not only for designing more effective AMF inhibitors but also for studying general protein-inhibitor interactions.
Assuntos
Carboidratos/química , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/química , Fosfatos/química , Animais , Sítios de Ligação , Movimento Celular , Cristalografia por Raios X , Glucose-6-Fosfato Isomerase/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Relação Estrutura-AtividadeRESUMO
RNase L is activated by the binding of unusual 2',5'-linked oligoadenylates (2-5A) and acts as the effector enzyme of the 2-5A system, an interferon-induced anti-virus mechanism. Efforts have been made to understand the 2-5A binding mechanism, not only for scientific interests but also for the prospects that the understanding of such mechanisms lead to new remedies for viral diseases. We have recently elucidated the crystal structure of the 2-5A binding ankyrin repeat domain of human RNase L complexed with 2-5A. To determine the contributions of amino acid residues surrounding the 2-5A binding site, point mutants and a deletion mutant were designed based on the crystal structure. These mutant proteins were analyzed for their interaction with 2-5A using a steady-state fluorescence technique. In addition, full-length RNase L mutants were tested for their activation by 2-5A. The results reveal that pi-pi stacking interactions of Trp60 and Phe126, electrostatic interactions of Lys89 and Arg155, and hydrogen bonding by Glu131 make crucial contributions to 2-5A binding. It was also found that the crystal structure of the ankyrin repeat domain L.2-5A complex accurately portrays the 2-5A binding mode in full-length RNase L.
Assuntos
Nucleotídeos de Adenina/química , Endorribonucleases/genética , Oligorribonucleotídeos/química , Arginina/química , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Endorribonucleases/química , Ativação Enzimática , Deleção de Genes , Vetores Genéticos , Glutationa Transferase/metabolismo , Humanos , Ligação de Hidrogênio , Lisina/química , Modelos Químicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oligonucleotídeos/química , Fenilalanina/química , Ligação Proteica , Estrutura Terciária de Proteína , Triptofano/químicaRESUMO
S-adenosylhomocysteine hydrolase is a prospective target for developing new anti-malarial drugs. Inhibition of the hydrolase results in an anti-cellular effect due to the suppression of adenosylmethionine-dependent transmethylations. Based on the crystal structure of Plasmodium falciparum S-adenosylhomocysteine hydrolase which we have determined recently, we performed mutational analyses on P. falciparum and human enzymes. Cys59 and Ala84 of the parasite enzyme, and the equivalent residues on the human enzyme, Thr60 and Gln85, were examined. Mutations of Cys59 and Thr60 caused dramatic impact on inhibition by 2-fluoronoraristeromycin without significant effect both on its kinetic parameters and on inhibition constant against noraristeromycin. In addition, the impact was independent from the electronegativity of the side chain of the substituting residue. These results showed that steric hindrance between a functional group at the 2-position of an adenine nucleoside inhibitor and Thr60 of the human enzyme, not an electrostatic effect, contributed to inhibitor selectivity.
Assuntos
Adenosina/análogos & derivados , Adenosil-Homocisteinase/genética , Adenosil-Homocisteinase/metabolismo , Plasmodium falciparum/enzimologia , Adenosina/farmacologia , Adenosil-Homocisteinase/química , Substituição de Aminoácidos , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Plasmodium falciparum/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade EstáticaRESUMO
Phosphoglucose isomerase (PGI; EC 5.3.1.9) is a cytosolic housekeeping enzyme of the sugar metabolism pathways that plays a key role in both glycolysis and gluconeogenesis. PGI is a multifunctional dimeric protein that extracellularly acts as a cytokine with properties that include autocrine motility factor (AMF)-eliciting mitogenic, motogenic, and differentiation functions, and PGI has been implicated in tumor progression and metastasis. Little is known of the biochemical regulation of PGI/AMF activities, although it is known that human PGI/AMF is phosphorylated at Ser(185) by protein kinase CK2 (CK2); however, the physiological significance of this phosphorylation is unknown. Thus, by site-directed mutagenesis, we substituted Ser(185) with aspartic acid (S185D) or glutamic acid (S185E), which introduces a negative charge and conformational changes that mimic phosphorylation. A Ser-to-Ala mutant protein (S185A) was generated to abolish phosphorylation. Biochemical analyses revealed that the phosphorylation mutant proteins of PGI exhibited decreased enzymatic activity, whereas the S185A mutant PGI protein retained full enzymatic activity. PGI phosphorylation by CK2 also led to down-regulation of enzymatic activity. Furthermore, CK2 knockdown by RNA interference was associated with up-regulation of cellular PGI enzymatic activity. The three recombinant mutant proteins exhibited indistinguishable cytokine activity and receptor-binding affinities compared with the wild-type protein. In both in vitro and in vivo assays, the wild-type and S185A mutant proteins underwent active species dimerization, whereas both the S185D and S185E mutant proteins also formed tetramers. These results demonstrate that phosphorylation affects the allosteric kinetic properties of the enzyme, resulting in a less active form of PGI, whereas non-phosphorylated protein species retain cytokine activity. The process by which phosphorylation modulates the enzymatic activity of PGI thus has an important implication for the understanding of the biological regulation of this key glucose metabolism-regulating enzyme.
Assuntos
Caseína Quinase II/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucose-6-Fosfato Isomerase/biossíntese , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/farmacologia , Citocinas/metabolismo , Citosol/metabolismo , Dimerização , Progressão da Doença , Relação Dose-Resposta a Droga , Regulação para Baixo , Glucose/metabolismo , Glicólise , Humanos , Cinética , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Metástase Neoplásica , Fosforilação , Conformação Proteica , Estrutura Secundária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Serina/química , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
Ribonuclease L (RNase L) is implicated in both the molecular mechanisms of interferon action and the fundamental control of RNA stability in mammalian cells. RNase L is catalytically active only after binding an unusual activator molecule containing a 5'-phosphorylated 2',5'-linked oligoadenylate, [(pp)p(A2'p5')(n)A] (2-5A), in the N-terminal half. Here we report the crystal structure of the N-terminal ankyrin repeat domain (ANK) of human RNase L complexed with the activator 2-5A. The ANK folds into eight ankyrin repeat elements and forms an extended curved structure with a concave surface. The 2-5A molecule is accommodated in the concavity and interacts with ankyrin repeats 2 to 4. Two structurally equivalent 2-5A binding motifs are found at repeats 2 and 4. The molecular basis for 2-5A recognition by RNase L is essential for designing stable 2-5As with a high likelihood of activating RNase L.
Assuntos
Nucleotídeos de Adenina/química , Repetição de Anquirina , Endorribonucleases/química , Oligorribonucleotídeos/química , Cristalografia , Humanos , Estrutura Terciária de ProteínaRESUMO
Mouse autocrine motility factor (mAMF), a tumour-secreted cytokine that stimulates cell migration in vitro and metastasis in vivo, has been crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 69.97, b = 115.88, c = 73.27 A, beta = 101.76 degrees . There are two subunits (one dimer) per asymmetric unit. Complexes with four-, five- and six-carbon carbohydrate phosphate inhibitors have also been crystallized. The crystals diffract to at least 1.8 A resolution and are suitable for X-ray structure analyses at high resolution.
Assuntos
Glucose-6-Fosfato Isomerase/química , Animais , Cristalização , Cristalografia por Raios X , Expressão Gênica , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/isolamento & purificação , CamundongosRESUMO
The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.
Assuntos
Adenosil-Homocisteinase/química , Plasmodium falciparum/enzimologia , Adenosil-Homocisteinase/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
An interferon-induced endoribonuclease, ribonuclease L (RNase L), is implicated in both the molecular mechanism of action of interferon and the fundamental control of RNA stability in mammalian cells. RNase L is catalytically active only after binding to an unusual activator molecule containing a 5'-phosphorylated 2',5'-linked oligoadenylate (2-5A), in the N-terminal half. Here, we report the crystal structure of the N-terminal ankyrin repeat domain (ANK) of human RNase L complexed with the activator 2-5A. This is the first structural view of an ankyrin repeat structure directly interacting with a nucleic acid, rather than with a protein. The ANK domain folds into eight ankyrin repeat elements and forms an extended curved structure with a concave surface. The 2-5A molecule is accommodated at a concave site and directly interacts with ankyrin repeats 2-4. Interestingly, two structurally equivalent 2-5A binding motifs are found at repeats 2 and 4. The structural basis for 2-5A recognition by ANK is essential for designing stable 2-5As with a high likelihood of activating RNase L.