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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed ß7-ß8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 µM). Inhibitor 6c displayed tighter binding (Kd â¼ 0.3 µM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.
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Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge in vivo. We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C' loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs.
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Vírus da Encefalite Transmitidos por Carrapatos , Nanopartículas , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Epitopos , CamundongosRESUMO
Change history: In the HTML version of this Letter, Extended Data Fig. 4 incorrectly corresponded to Fig. 4 (the PDF version of the figure was correct). This has been corrected online.
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Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes.
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Antivirais/metabolismo , Citidina Trifosfato/metabolismo , Genoma Humano/genética , Proteínas/genética , Proteínas/metabolismo , Terminação da Transcrição Genética , Animais , Antivirais/química , Chlorocebus aethiops , Citidina Trifosfato/biossíntese , Citidina Trifosfato/química , Células HEK293 , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Ribonucleotídeos , Especificidade por Substrato , Células Vero , Zika virus/enzimologia , Zika virus/metabolismoRESUMO
S-Adenosyl-l-methionine (AdoMet) is synthesized by the MAT2A isozyme of methionine adenosyltransferase in most human tissues and in cancers. Its contribution to epigenetic control has made it a target for anticancer intervention. A recent kinetic isotope effect analysis of MAT2A demonstrated a loose nucleophilic transition state. Here we show that MAT2A has a sequential mechanism with a rate-limiting step of formation of AdoMet, followed by rapid hydrolysis of the ß-γ bond of triphosphate, and rapid release of phosphate and pyrophosphate. MAT2A catalyzes the slow hydrolysis of both ATP and triphosphate in the absence of other reactants. Positional isotope exchange occurs with 18O as the 5'-oxygen of ATP. Loss of the triphosphate is sufficiently reversible to permit rotation and recombination of the α-phosphoryl group of ATP. Adenosine (α-ß or ß-γ)-imido triphosphates are slow substrates, and the respective imido triphosphates are inhibitors. The hydrolytically stable (α-ß, ß-γ)-diimido triphosphate (PNPNP) is a nanomolar inhibitor. The MAT2A protein structure is highly stabilized against denaturation by binding of PNPNP. A crystal structure of MAT2A with 5'-methylthioadenosine and PNPNP shows the ligands arranged appropriately in the ATP binding site. Two magnesium ions chelate the α- and γ-phosphoryl groups of PNPNP. The ß-phosphoryl oxygen is in contact with an essential potassium ion. Imidophosphate derivatives provide contact models for the design of catalytic site ligands for MAT2A.
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Trifosfato de Adenosina/metabolismo , Difosfatos/metabolismo , Inibidores Enzimáticos/farmacologia , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/metabolismo , Polifosfatos/metabolismo , S-Adenosilmetionina/farmacologia , Sítios de Ligação , Humanos , Hidrólise , Cinética , Conformação ProteicaRESUMO
Human methionine adenosyltransferase MAT2A provides S-adenosyl-l-methionine (AdoMet) for methyl-transfer reactions. Epigenetic methylations influence expression patterns in development and in cancer. Transition-state analysis and kinetic studies have described the mechanism of AdoMet and triphosphate formation at the catalytic site. Hydrolysis of triphosphate to pyrophosphate and phosphate by MAT2A is required for product release and proceeds through a second chemical transition state. Crystal structures of MAT2A with analogues of AdoMet and pyrophosphate were obtained in the presence of Mg2+, Al3+, and F-. MgF3- is trapped as a PO3- mimic in a structure with malonate filling the pyrophosphate site. NMR demonstrates that MgF3- and AlF30 are bound by MAT2A as mimics of the departing phosphoryl group. Crystallographic analysis reveals a planar MgF3- acting to mimic a phosphoryl (PO3-) leaving group. The modeled transition state with PO3- has the phosphorus atom sandwiched symmetrically and equidistant (approximately 2 Å) between a pyrophosphate oxygen and the water nucleophile. A catalytic site arginine directs the nucleophilic water to the phosphoryl leaving group. The catalytic geometry of the transition-state reconstruction predicts a loose transition state with characteristics of symmetric nucleophilic displacement.
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Biocatálise , Metionina Adenosiltransferase/metabolismo , Polifosfatos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Humanos , Hidrólise , Metionina Adenosiltransferase/química , Modelos Químicos , Polifosfatos/química , Ligação Proteica , Água/metabolismoRESUMO
PURPOSE: MR spectroscopy (MRS) can improve diagnosis and follow treatment in cancer. However, no study has yet reported application of in vivo (1)H-MRS in malignant pancreatic lesions. This study quantitatively determined whether in vivo (1)H-MRS on multiple endocrine neoplasia type 1 (Men1) conditional knockout (KO) mice and their wild type (WT) littermates could detect differences in total choline (tCho) levels between tumor and control pancreas. METHODS: Relative tCho levels in pancreatic tumors or pancreata from KO and WT mice were determined using in vivo (1)H-MRS at 9.4 T. The levels of Cho-containing compounds were also quantified using in vitro (1)H-NMR on extracts of pancreatic tissues from KO and WT mice, respectively, and on extracts of pancreatic tissues from patients with pancreatic neuroendocrine tumors (PNETs). RESULTS: tCho levels measured by in vivo (1)H-MRS were significantly higher in PNETs from KO mice compared to the normal pancreas from WT mice. The elevated choline-containing compounds were also identified in pancreatic tumors from KO mice and tissues from patients with PNETs via in vitro (1)H-NMR. CONCLUSION: These results indicate the potential use of tCho levels estimated via in vivo (1)H-MRS in differentiating malignant pancreatic tumors from benign tumors.
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Imageamento por Ressonância Magnética/métodos , Neoplasia Endócrina Múltipla Tipo 1/química , Neoplasia Endócrina Múltipla Tipo 1/diagnóstico , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/diagnóstico , Animais , Colina/análise , Colina/química , Colina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , Neoplasia Endócrina Múltipla Tipo 1/patologia , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/genéticaRESUMO
Isocitrate lyase (ICL) catalyzes the reversible retro-aldol cleavage of isocitrate to generate glyoxylate and succinate. ICL is the first enzyme of the glyoxylate shunt, which allows for the anaplerosis of citric acid cycle intermediates under nutrient limiting conditions. In Mycobacterium tuberculosis, the source of ICL for these studies, ICL is vital for the persistence phase of the bacterium's life cycle. Solvent kinetic isotope effects (KIEs) in the direction of isocitrate cleavage ((D2O)V = 2.0 ± 0.1, and (D2O)[V/K(isocitrate)] = 2.2 ± 0.3) arise from the initial deprotonation of the C2 hydroxyl group of isocitrate or the protonation of the aci-acid of the succinate product of the isocitrate aldol cleavage by a solvent-derived proton. This KIE suggested that an equilibrium mixture of all protiated isocitrate, glyoxylate, and succinate prepared in D2O would undergo transient changes in equilibrium concentrations as a result of the solvent KIE and solvent-derived deuterium incorporation into both succinate and isocitrate. No change in the isotopic composition of glyoxylate was expected or observed. We have directly monitored the changing concentrations of all isotopic species of all reactants and products using a combination of nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Continuous monitoring of glyoxylate by ¹H NMR spectroscopy shows a clear equilibrium perturbation in D2O. The final equilibrium isotopic composition of reactants in D2O revealed dideuterated succinate, protiated glyoxylate, and monodeuterated isocitrate, with the transient appearance and disappearance of monodeuterated succinate. A model for the equilibrium perturbation of substrate species and their time-dependent isotopic composition is presented.
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Proteínas de Bactérias/metabolismo , Isocitrato Liase/metabolismo , Isocitratos/metabolismo , Modelos Moleculares , Água/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Simulação por Computador , Medição da Troca de Deutério , Glioxilatos/química , Glioxilatos/metabolismo , Hidrólise , Isocitrato Liase/química , Isocitrato Liase/genética , Isocitratos/química , Cinética , Espectrometria de Massas , Mycobacterium tuberculosis/enzimologia , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ácido Succínico/química , Ácido Succínico/metabolismoRESUMO
Toxins TcdA and TcdB from Clostridioides difficile glucosylate human colon Rho GTPases. TcdA and TcdB glucosylation of RhoGTPases results in cytoskeletal changes, causing cell rounding and loss of intestinal integrity. Clostridial toxins TcdA and TcdB are proposed to catalyze glucosylation of Rho GTPases with retention of stereochemistry from UDP-glucose. We used kinetic isotope effects to analyze the mechanisms and transition-state structures of the glucohydrolase and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase glucosylation with retention of stereochemistry, while hydrolysis of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic analysis revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an SNi mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mechanical calculations revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an SNi mechanism with the formation of a distinct oxocarbenium phosphate ion pair transition state where the glycosidic bond to the UDP leaving group breaks prior to attack of the threonine nucleophile from Rho GTPase.
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Toxinas Bacterianas , Clostridioides difficile , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Glucose , Glucosiltransferases/metabolismo , Humanos , Cinética , Fosfatos , Toxina Tetânica , Treonina , Uridina Difosfato Glucose , Proteínas rho de Ligação ao GTPRESUMO
Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) is essential for purine salvage of hypoxanthine into parasite purine nucleotides. Transition state analogue inhibitors of PfHGXPRT are characterized by kinetic analysis, thermodynamic parameters, and X-ray crystal structures. Compound 1, 9-deazaguanine linked to an acyclic ribocation phosphonate mimic, shows a kinetic Ki of 0.5 nM. Isothermal titration calorimetry (ITC) experiments of 1 binding to PfHGXPRT reveal enthalpically driven binding with negative cooperativity for the binding of two inhibitor molecules in the tetrameric enzyme. Crystal structures of 1 bound to PfHGXPRT define the hydrogen bond and ionic contacts to complement binding thermodynamics. Dynamics of ribosyl transfer from 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) to hypoxanthine were examined by 18O isotope exchange at the bridging phosphoryl oxygen of PRPP pyrophosphate. Rotational constraints or short transition state lifetimes prevent torsional rotation and positional isotope exchange of bridging to nonbridging oxygen in the α-pyrophosphoryl group. Thermodynamic analysis of the transition state analogue and magnesium pyrophosphate binding reveal random and cooperative binding to PfHGXPRT, unlike the obligatory ordered reaction kinetics reported earlier for substrate kinetics.
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Difosfatos , Plasmodium falciparum , Cinética , Isótopos , Oxigênio , HipoxantinasRESUMO
Zika virus (ZIKV) is a flavivirus that can cause severe disease, but there are no approved treatments or vaccines. A complication for flavivirus vaccine development is the potential of immunogens to enhance infection via antibody-dependent enhancement (ADE), a process mediated by poorly neutralizing and cross-reactive antibodies. Thus, there is a great need to develop immunogens that minimize the potential to elicit enhancing antibodies. Here we utilized structure-based protein engineering to develop "resurfaced" (rs) ZIKV immunogens based on E glycoprotein domain III (ZDIIIs), in which epitopes bound by variably neutralizing antibodies were masked by combinatorial mutagenesis. We identified one resurfaced ZDIII immunogen (rsZDIII-2.39) that elicited a protective but immune-focused response. Compared to wild type ZDIII, immunization with resurfaced rsZDIII-2.39 protein nanoparticles produced fewer numbers of ZIKV EDIII antigen-reactive B cells and elicited serum that had a lower magnitude of induced ADE against dengue virus serotype 1 (DENV1) Our findings enhance our understanding of the structural and functional determinants of antibody protection against ZIKV.
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Vírus da Dengue , Nanopartículas , Infecção por Zika virus , Zika virus , Anticorpos Neutralizantes , Anticorpos Antivirais , Vírus da Dengue/química , Humanos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Infecção por Zika virus/prevenção & controleRESUMO
The EntE enzyme, involved in the synthesis of the iron siderophore enterobactin, catalyzes the adenylation of 2,3-dihydroxybenzoic acid, followed by its transfer to the phosphopantetheine arm of holo-EntB, an aryl carrier protein. In the absence of EntB, EntE catalyzes the formation of Ap(4)A, a molecule that is implicated in regulating cell division during oxidative stress. We propose that the expression of EntE during iron starvation produces Ap(4)A to slow growth until intracellular iron stores can be restored.
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Fosfatos de Dinucleosídeos/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Ligases/metabolismo , Trifosfato de Adenosina/metabolismo , Biocatálise , Cromatografia Líquida de Alta Pressão , Fosfatos de Dinucleosídeos/análise , Retroalimentação Fisiológica/fisiologia , Hidrolases/metabolismo , Hidroxibenzoatos/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Complexos Multienzimáticos/metabolismo , Ácido Salicílico/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Riboflavin is biosynthesized by most microorganisms and plants, while mammals depend entirely on the absorption of this vitamin from the diet to meet their metabolic needs. Therefore, riboflavin biosynthesis appears to be an attractive target for drug design, since appropriate inhibitors of the pathway would selectively target the microorganism. We have cloned and solubly expressed the bifunctional ribD gene from Escherichia coli, whose three-dimensional structure was recently determined. We have demonstrated that the rate of deamination (370 min (-1)) exceeds the rate of reduction (19 min (-1)), suggesting no channeling between the two active sites. The reductive ring opening reaction occurs via a hydride transfer from the C 4- pro-R hydrogen of NADPH to C'-1 of ribose and is the rate-limiting step in the overall reaction, exhibiting a primary kinetic isotope effect ( (D) V) of 2.2. We also show that the INH-NADP adduct, one of the active forms of the anti-TB drug isoniazid, inhibits the E. coli RibD. On the basis of the observed patterns of inhibition versus the two substrates, we propose that the RibD-catalyzed reduction step follows a kinetic scheme similar to that of its structural homologue, DHFR.
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Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Nucleotídeo Desaminases/química , Riboflavina/biossíntese , Riboflavina/química , Desidrogenase do Álcool de Açúcar/química , Catálise , Desaminação , Medição da Troca de Deutério , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , NADP/química , NADP/metabolismo , Nucleotídeo Desaminases/genética , Nucleotídeo Desaminases/metabolismo , Oxirredução , Pentosefosfatos/química , Pentosefosfatos/metabolismo , Estrutura Terciária de Proteína/genética , Bases de Schiff , Solventes , Especificidade por Substrato/genética , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/metabolismoRESUMO
A new mechanistic principle for reporting the phosphorylation of tyrosine is described, which should prove applicable to even the most fastidious of protein tyrosine kinases, as demonstrated by the acquisition of a fluorescent sensor for the extraordinarily demanding anaplastic lymphoma kinase.
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Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Antineoplásicos/química , Corantes Fluorescentes , Estrutura Molecular , Fosforilação , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/química , Receptores Proteína Tirosina QuinasesRESUMO
INTRODUCTION: There is a lot of interest towards creating therapies and vaccines for Bacillus anthracis, a bacterium which causes anthrax in humans and which spores can be made into potent biological weapons. Systemic injection of lethal factor (LF), edema factor (EF) and protective antigen (PA) in mice produces toxicity, and this protocol is commonly used to investigate the efficacy of specific antibodies in passive protection and vaccine studies. Availability of toxins labeled with imageable radioisotopes would allow to demonstrate their tissue distribution after intravenous injection at toxin concentration that are below pharmacologically significant to avoid masking by toxic effects. METHODS: LF, EF and PA were radiolabeled with (188)Re and (99m)Tc, and their performance in vitro was evaluated by macrophages and Chinese hamster ovary cells toxicity assays and by binding to macrophages. Scintigraphic imaging and biodistribution of intravenously (IV) injected (99m)Tc-and (123)I-labeled toxins was performed in BALB/c mice. RESULTS: Radiolabeled toxins preserved their biological activity. Scatchard-type analysis of the binding of radiolabeled PA to the J774.16 macrophage-like cells revealed 6.6 x 10(4) binding sites per cell with a dissociation constant of 6.7 nM. Comparative scintigraphic imaging of mice injected intravenously with either (99m)Tc-or (123)I-labeled PA, EF and LF toxins demonstrated similar biodistribution patterns with early localization of radioactivity in the liver, spleen, intestines and excretion through kidneys. The finding of renal excretion shortly after IV injection strongly suggests that toxins are rapidly degraded which could contribute to the variability of mouse toxigenic assays. Biodistribution studies confirmed that all three toxins concentrated in the liver and the presence of high levels of radioactivity again implied rapid degradation in vivo. CONCLUSIONS: The availability of (188)Re and (99m)Tc-labeled PA, LF and EF toxins allowed us to confirm the number of PA binding sites per cell, to provide an estimate of the dissociation constant of PA for its receptor and to demonstrate tissue distribution of toxins in mice after intravenous injection.
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Toxinas Bacterianas/farmacocinética , Radioisótopos , Rênio , Animais , Antígenos de Bactérias , Células CHO , Cricetinae , Cricetulus , Feminino , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos BALB C , Tecnécio , Distribuição TecidualRESUMO
The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the â¼120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-of-pearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.
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Glicoproteínas de Membrana/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Adesão Celular , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
The conformation of the ATP synthase c-subunit and the pKa of its essential E54 residue were characterized in alkaliphilic Bacillus pseudofirmus OF4. The c-subunit folds as a helix-loop-helix, with inter-helical contacts demonstrated by paramagnetic relaxation effects. The E54 pKa of 7.7 is significantly higher than in non-alkaliphiles, which likely prevents proton loss from the c-rotor at high pH. The E54 pKa was unchanged in a mutant, cP51A, that has a severe ATP synthesis defect at high pH only. cP51 must have some structural role that accounts for the mutant defect, such as different subunit-subunit interactions at high pH.
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Bacillus/enzimologia , Proteínas de Bactérias/química , Ácido Glutâmico/química , ATPases Mitocondriais Próton-Translocadoras/química , Conformação Proteica , Subunidades Proteicas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Concentração de Íons de Hidrogênio , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , PrótonsRESUMO
Purine nucleoside phosphorylase (PNP) is a target for leukemia, gout, and autoimmune disorders. Dynamic motion of catalytic site loops has been implicated in catalysis, but experimental evidence was lacking. We replaced catalytic site groups His257 or His64 with 6-fluoro-tryptophan (6FW) as site-specific NMR probes. Conformational adjustments in the 6FW-His257-helical and His64-6FW-loop regions were characterized in PNP phosphate-bound enzyme and in complexes with catalytic site ligands, including transition state analogs. Chemical shift and line-shape changes associated with these complexes revealed dynamic coexistence of several conformational states in these regions in phosphate-bound enzyme and altered or single conformations in other complexes. These conformations were also characterized by X-ray crystallography. Specific (19)F-Trp labels and X-ray crystallography provide multidimensional characterization of conformational states for free, catalytic, and inhibited complexes of human PNP.