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1.
Blood ; 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32040546

RESUMO

In gene therapy with human hematopoietic stem and progenitor cells (HSPCs), each gene-corrected cell and its progeny are marked in a unique way by the integrating vector. This feature enables lineages to be tracked by sampling blood cells and using DNA sequencing to identify the vector integration sites. Here, we studied five cell lineages (granulocytes, monocytes, T cells, B cells, and natural killer cells) in patients having undergone HSPC gene therapy for Wiskott-Aldrich syndrome or beta hemoglobinopathies. We found that the estimated minimum number of active, repopulating HSPCs (which ranged from 2,000 to 50,000) was correlated with the number of HSPCs per kg infused. We sought to quantify the lineage output and dynamics of gene-modified clones; this is usually challenging because of (i) sparse sampling of the various cell types during the analytical procedure, (ii) contamination during cell isolation, and (iii) different levels of vector marking in the various lineages. We therefore measured the residual contamination and corrected our statistical models accordingly, in order to provide a rigorous analysis of the HSPC lineage output. A cluster analysis of the HSPC lineage output highlighted the existence of several stable, distinct differentiation programs, including myeloid-dominant, lymphoid-dominant and balanced cell subsets. Our study evidenced the heterogeneous nature of the cell lineage output from HSPCs, and provided methods for analyzing these complex data.

2.
Nat Med ; 26(2): 200-206, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31988463

RESUMO

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.

3.
J Clin Invest ; 130(2): 673-685, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31845905

RESUMO

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) provide an effective treatment for pediatric acute lymphoblastic leukemia but are less effective for chronic lymphocytic leukemia (CLL), focusing attention on improving efficacy. CART19 harbor an engineered receptor, which is delivered through lentiviral vector integration, thereby marking cell lineages and modifying the cellular genome by insertional mutagenesis. We recently reported that vector integration within the host TET2 gene was associated with CLL remission. Here, we investigated clonal population structure and therapeutic outcomes in another 39 patients by high-throughput sequencing of vector-integration sites. Genes at integration sites enriched in responders were commonly found in cell-signaling and chromatin modification pathways, suggesting that insertional mutagenesis in these genes promoted therapeutic T cell proliferation. We also developed a multivariate model based on integration-site distributions and found that data from preinfusion products forecasted response in CLL successfully in discovery and validation cohorts and, in day 28 samples, reported responders to CLL therapy with high accuracy. These data clarify how insertional mutagenesis can modulate cell proliferation in CART19 therapy and how data on integration-site distributions can be linked to treatment outcomes.

4.
Methods Enzymol ; 615: 453-475, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30638538

RESUMO

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand-receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand-receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Ligantes , Receptores de Superfície Celular/metabolismo , Cromatografia de Afinidade , Cristalografia por Raios X , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica
5.
Genome Med ; 10(1): 70, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30261899

RESUMO

BACKGROUND: Mutation of the IL2RG gene results in a form of severe combined immune deficiency (SCID-X1), which has been treated successfully with hematopoietic stem cell gene therapy. SCID-X1 gene therapy results in reconstitution of the previously lacking T cell compartment, allowing analysis of the roles of T cell immunity in humans by comparing before and after gene correction. METHODS: Here we interrogate T cell reconstitution using four forms of high throughput analysis. (1) Estimation of the numbers of transduced progenitor cells by monitoring unique positions of integration of the therapeutic gene transfer vector. (2) Estimation of T cell population structure by sequencing of the recombined T cell receptor (TCR) beta locus. (3) Metagenomic analysis of microbial populations in oropharyngeal, nasopharyngeal, and gut samples. (4) Metagenomic analysis of viral populations in gut samples. RESULTS: Comparison of progenitor and mature T cell populations allowed estimation of a minimum number of cell divisions needed to generate the observed populations. Analysis of microbial populations showed the effects of immune reconstitution, including normalization of gut microbiota and clearance of viral infections. Metagenomic analysis revealed enrichment of genes for antibiotic resistance in gene-corrected subjects relative to healthy controls, likely a result of higher healthcare exposure. CONCLUSIONS: This multi-omic approach enables the characterization of multiple effects of SCID-X1 gene therapy, including T cell repertoire reconstitution, estimation of numbers of cell divisions between progenitors and daughter T cells, normalization of the microbiome, clearance of microbial pathogens, and modulations in antibiotic resistance gene levels. Together, these results quantify several aspects of the long-term efficacy of gene therapy for SCID-X1. This study includes data from ClinicalTrials.gov numbers NCT01410019, NCT01175239, and NCT01129544.


Assuntos
Terapia Genética , Microbiota , Linfócitos T/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Divisão Celular , Pré-Escolar , Regiões Determinantes de Complementaridade/genética , Humanos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/microbiologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/virologia
6.
Nature ; 558(7709): 307-312, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29849141

RESUMO

Cancer immunotherapy based on genetically redirecting T cells has been used successfully to treat B cell malignancies1-3. In this strategy, the T cell genome is modified by integration of viral vectors or transposons encoding chimaeric antigen receptors (CARs) that direct tumour cell killing. However, this approach is often limited by the extent of expansion and persistence of CAR T cells4,5. Here we report mechanistic insights from studies of a patient with chronic lymphocytic leukaemia treated with CAR T cells targeting the CD19 protein. Following infusion of CAR T cells, anti-tumour activity was evident in the peripheral blood, lymph nodes and bone marrow; this activity was accompanied by complete remission. Unexpectedly, at the peak of the response, 94% of CAR T cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene disrupted the methylcytosine dioxygenase TET2 gene. Further analysis revealed a hypomorphic mutation in this patient's second TET2 allele. TET2-disrupted CAR T cells exhibited an epigenetic profile consistent with altered T cell differentiation and, at the peak of expansion, displayed a central memory phenotype. Experimental knockdown of TET2 recapitulated the potency-enhancing effect of TET2 dysfunction in this patient's CAR T cells. These findings suggest that the progeny of a single CAR T cell induced leukaemia remission and that TET2 modification may be useful for improving immunotherapies.


Assuntos
5-Metilcitosina/metabolismo , Antígenos CD19/imunologia , Dioxigenases/genética , Imunoterapia/métodos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Linfócitos T/imunologia , Linfócitos T/transplante , Transferência Adotiva , Idoso , Alelos , Diferenciação Celular , Ensaios Clínicos como Assunto , Células Clonais/citologia , Células Clonais/imunologia , Dioxigenases/metabolismo , Epigênese Genética , Células HEK293 , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Transgenes
7.
Mol Ther Methods Clin Dev ; 4: 17-26, 2017 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-28344988

RESUMO

Analysis of sites of newly integrated DNA in cellular genomes is important to several fields, but methods for analyzing and visualizing these datasets are still under development. Here, we describe tools for data analysis and visualization that take as input integration site data from our INSPIIRED pipeline. Paired-end sequencing allows inference of the numbers of transduced cells as well as the distributions of integration sites in target genomes. We present interactive heatmaps that allow comparison of distributions of integration sites to genomic features and that support numerous user-defined statistical tests. To summarize integration site data from human gene therapy samples, we developed a reproducible report format that catalogs sample population structure, longitudinal dynamics, and integration frequency near cancer-associated genes. We also introduce a novel summary statistic, the UC50 (unique cell progenitors contributing the most expanded 50% of progeny cell clones), which provides a single number summarizing possible clonal expansion. Using these tools, we characterize ongoing longitudinal characterization of a patient from the first trial to treat severe combined immunodeficiency-X1 (SCID-X1), showing successful reconstitution for 15 years accompanied by persistence of a cell clone with an integration site near the cancer-associated gene CCND2. Software is available at https://github.com/BushmanLab/INSPIIRED.

8.
Mol Ther Methods Clin Dev ; 4: 39-49, 2017 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-28344990

RESUMO

Integration of new DNA into cellular genomes mediates replication of retroviruses and transposons; integration reactions have also been adapted for use in human gene therapy. Tracking the distributions of integration sites is important to characterize populations of transduced cells and to monitor potential outgrow of pathogenic cell clones. Here, we describe a pipeline for quantitative analysis of integration site distributions named INSPIIRED (integration site pipeline for paired-end reads). We describe optimized biochemical steps for site isolation using Illumina paired-end sequencing, including new technology for suppressing recovery of unwanted contaminants, then software for alignment, quality control, and management of integration site sequences. During library preparation, DNAs are broken by sonication, so that after ligation-mediated PCR the number of ligation junction sites can be used to infer abundance of gene-modified cells. We generated integration sites of known positions in silico, and we describe optimization of sample processing parameters refined by comparison to truth. We also present a novel graph-theory-based method for quantifying integration sites in repeated sequences, and we characterize the consequences using synthetic and experimental data. In an accompanying paper, we describe an additional set of statistical tools for data analysis and visualization. Software is available at https://github.com/BushmanLab/INSPIIRED.

9.
Mol Cell Proteomics ; 16(2): 194-212, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27923875

RESUMO

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene.


Assuntos
Fígado/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteoma/análise , Proteômica/métodos , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Bases de Dados de Proteínas , Humanos , Lactente , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/metabolismo , Espectrometria de Massas , Mutação , Doenças Neurodegenerativas/genética , Ratos , Análise de Sequência de DNA , Frações Subcelulares/metabolismo
10.
Protein Sci ; 25(7): 1299-307, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26873166

RESUMO

Design of polar interactions is a current challenge for protein design. The de novo designed protein Top7, like almost all designed proteins, has an entirely nonpolar core. Here we describe the replacing of a sizable fraction (5 residues) of this core with a designed polar hydrogen bond network. The polar core design is expressed at high levels in E. coli, has a folding free energy of 10 kcal/mol, and retains the multiphasic folding kinetics of the original Top7. The NMR structure of the design shows that conformations of three of the five residues, and the designed hydrogen bonds between them, are very close to those in the design model. The remaining two residues, which are more solvent exposed, sample a wide range of conformations in the NMR ensemble. These results show that hydrogen bond networks can be designed in protein cores, but also highlight challenges that need to be overcome when there is competition with solvent.


Assuntos
Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Proteínas/química , Cristalografia por Raios X , Escherichia coli/genética , Ligações de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas/metabolismo
11.
Nature ; 529(7586): 358-363, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26760206

RESUMO

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyse the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.


Assuntos
Códon/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Genes Sintéticos/genética , Meia-Vida , Cinética , Modelos Logísticos , Modelos Genéticos , Dados de Sequência Molecular , Razão de Chances , Elongação Traducional da Cadeia Peptídica , Dobramento de RNA , Estabilidade de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Transcrição Genética/genética , Proteínas Virais/metabolismo
12.
Protein Sci ; 25(1): 30-45, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26293815

RESUMO

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.


Assuntos
Cristalografia por Raios X , Bases de Dados de Proteínas , Ressonância Magnética Nuclear Biomolecular , Modelos Moleculares , Conformação Proteica , Proteínas/química
13.
Struct Dyn ; 2(4): 041710, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26798809

RESUMO

Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization.

14.
Biomol NMR Assign ; 9(1): 135-138, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24722902

RESUMO

The 500 kDa protein plectin is essential for the cytoskeletal organization of most mammalian cells and it is up-regulated in some types of cancer. Here, we report nearly complete sequence-specific polypeptide backbone, (13)C(ß) and methyl group resonance assignments for 24 kDa human plectin(4403-4606) containing the C-terminal plectin repeat domain 6.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Plectina/química , Humanos , Estrutura Terciária de Proteína
15.
Proc Natl Acad Sci U S A ; 111(44): E4697-705, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25339443

RESUMO

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1-9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Ubiquinona/biossíntese , Animais , Proteínas de Transporte/genética , Cristalografia por Raios X , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Mutantes , Proteínas Mitocondriais/genética , Estrutura Terciária de Proteína , Ubiquinona/genética
18.
J Struct Funct Genomics ; 15(4): 209-14, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24989974

RESUMO

High-quality solution NMR structures of immunoglobulin-like domains 7 and 12 from human obscurin-like protein 1 were solved. The two domains share 30% sequence identity and their structures are, as expected, rather similar. The new structures contribute to structural coverage of human cancer associated proteins. Mutations of Arg 812 in domain 7 cause the rare 3-M syndrome, and this site is located in a surface area predicted to be involved in protein-protein interactions.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas de Neoplasias/química , Neoplasias/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína
19.
J Struct Funct Genomics ; 15(4): 201-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24941917

RESUMO

High-quality solution NMR structures of three homeodomains from human proteins ALX4, ZHX1 and CASP8AP2 were solved. These domains were chosen as targets of a biomedical theme project pursued by the Northeast Structural Genomics Consortium. This project focuses on increasing the structural coverage of human proteins associated with cancer.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação a DNA/química , Proteínas de Homeodomínio/química , Proteínas de Neoplasias/química , Neoplasias/química , Fatores de Transcrição/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína
20.
FEBS J ; 281(6): 1613-1628, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24456211

RESUMO

UNLABELLED: Cytosolic nucleotidase II (cN-II) from Legionella pneumophila (Lp) catalyzes the hydrolysis of GMP and dGMP displaying sigmoidal curves, whereas catalysis of IMP hydrolysis displayed a biphasic curve in the initial rate versus substrate concentration plots. Allosteric modulators of mammalian cN-II did not activate LpcN-II although GTP, GDP and the substrate GMP were specific activators. Crystal structures of the tetrameric LpcN-II revealed an activator-binding site at the dimer interface. A double mutation in this allosteric-binding site abolished activation, confirming the structural observations. The substrate GMP acting as an activator, partitioning between the allosteric and active site, is the basis for the sigmoidicity of the initial velocity versus GMP concentration plot. The LpcN-II tetramer showed differences in subunit organization upon activator binding that are absent in the activator-bound human cN-II structure. This is the first observation of a structural change induced by activator binding in cN-II that may be the molecular mechanism for enzyme activation. DATABASE: The coordinates and structure factors reported in this paper have been submitted to the Protein Data Bank under the accession numbers 2BDE and 4G63. The accession number of GMP complexed LpcN-II is 4OHF. STRUCTURED DIGITAL ABSTRACT: LpcN-II and LpcN-II bind by molecular sieving (View interaction) LpcN-II and LpcN-II bind by x-ray crystallography (View interaction) [Structured digital abstract was added on 5 March 2014 after original online publication].


Assuntos
5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Legionella pneumophila/enzimologia , 5'-Nucleotidase/genética , Regulação Alostérica , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Nucleotídeos de Desoxiguanina/metabolismo , Ativação Enzimática , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Legionella pneumophila/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Especificidade da Espécie , Especificidade por Substrato
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