RESUMO
The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogen-deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-B-dependent transcription exercised by GR in cells.
Assuntos
Proteínas de Ligação a DNA/química , Receptores Citoplasmáticos e Nucleares/química , Receptores de Esteroides/química , Fatores de Transcrição/química , Dicroísmo Circular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Dimerização , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Estrutura Secundária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Receptores de Glucocorticoides/química , Receptores de Esteroides/genética , Receptores de Esteroides/isolamento & purificação , Espalhamento a Baixo Ângulo , Soluções , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
The rat protein tyrosine phosphatase eta, rPTPeta, is a class I "classical" transmembrane RPTP, with an intracellular portion composed of a unique catalytic region. The rPTPeta and the human homolog DEP-1 are downregulated in rat and human neoplastic cells, respectively. However, the malignant phenotype is reverted after exogenous reconstitution of rPTPeta, suggesting that its function restoration could be an important tool for gene therapy of human cancers. Using small-angle x-ray scattering (SAXS) and biophysical techniques, we characterized the intracellular catalytic domain of rat protein tyrosine phosphatase eta (rPTPetaCD) in solution. The protein forms dimers in solution as confirmed by SAXS data analysis. The SAXS data also indicated that rPTPetaCD dimers are elongated and have an average radius of gyration of 2.65 nm and a D(max) of 8.5 nm. To further study the rPTPetaCD conformation in solution, we built rPTPetaCD homology models using as scaffolds the crystallographic structures of RPTPalpha-D1 and RPTPmicro-D1 dimers. These models were, then, superimposed onto ab initio low-resolution SAXS structures. The structural comparisons and sequence alignment analysis of the putative dimerization interfaces provide support to the notion that the rPTPetaCD dimer architecture is more closely related to the crystal structure of autoinhibitory RPTPalpha-D1 dimer than to the dimeric arrangement exemplified by RPTPmicro-D1. Finally, the characterization of rPTPetaCD by fluorescence anisotropy measurements demonstrates that the dimer dissociation is concentration dependent with a dissociation constant of 21.6 +/- 2.0 microM.
Assuntos
Cristalografia/métodos , Modelos Químicos , Modelos Moleculares , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/ultraestrutura , Anisotropia , Catálise , Simulação por Computador , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a ReceptoresRESUMO
The rat protein tyrosine phosphatase eta (rPTPeta) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPeta and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPeta, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPeta might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPeta was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 A resolution. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 A, and contains one molecule per asymmetric unit.
Assuntos
Subunidades Proteicas/química , Proteínas Tirosina Fosfatases/química , Animais , Domínio Catalítico , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Tirosina Fosfatases/isolamento & purificação , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Thyroid hormone nuclear receptors (TRs) bind to DNA and activate transcription as heterodimers with the retinoid X receptor (RXR) or as homodimers or monomers. RXR also binds to DNA and activates transcription as homodimers but can, in addition, self-associate into homotetramers in the absence of ligand and DNA templates. It is thought that homotetramer formation serves to sequester excess RXRs into an inactive pool within the cell. Here, we report systematic studies of the multimeric state of a recombinant human TRbeta1 truncation (hTRbeta1deltaAB) that encompasses the complete DNA binding domain and ligand binding domain in solution. Native gel electrophoresis, chemical crosslinking, gel filtration, and dynamic light scattering experiments reveal that hTRbeta1deltaAB forms a mixture of monomers, dimers, and tetramers. Like RXR, increasing protein concentration shifts the equilibrium between TR multimers toward tetramer formation, whereas binding of cognate thyroid hormone leads to dissociation of tetramers and increased formation of dimers. This work represents the first evidence that apo-hTRbeta1 forms homotetramers. The findings raise the possibility that tetramer formation provides an additional, and previously unsuspected, level of control of TR activity and that the capacity for homotetramer formation may be more widespread in the nuclear receptor family than previously thought.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Receptores beta dos Hormônios Tireóideos/química , Receptores beta dos Hormônios Tireóideos/metabolismo , Tri-Iodotironina/química , Tri-Iodotironina/metabolismo , Sequência de Aminoácidos/genética , DNA/química , DNA/fisiologia , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ligantes , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/fisiologia , Proteínas Recombinantes/genética , Receptores X de Retinoides , Soluções/química , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Receptor-like protein tyrosine phosphatases generally contain one or two conserved intracellular catalytic domains with a conserved sequence motif ([I/V]HCXAGXXR[S/T]G), a single transmembrane domain, and an external highly variable part. Here, we describe cloning of the intracellular catalytic domain of the rat protein tyrosine phosphatase eta (rPTPetaCD) into pET28a(+) vector, its expression in Escherichia coli, purification and initial characterization. The purification of His6-tagged rPTPetaCD to near homogeneity was achieved by a combination of affinity and size exclusion chromatography. The His-tag was subsequently removed by thrombin digestion. PhastGel IEF electrophoresis demonstrated that the isoelectric point of this 41 kDa His6-tag free recombinant protein was 7.3, which is just slightly higher than the theoretically predicted value of 7.2. To assess the functionality of the rPTPetaCD we used the pNPP hydrolysis assay and observed that the enzyme has a specific activity of 9 nmol/min/mug. The secondary structure and stability of the recombinant protein was also analyzed by circular dichroism and fluorescence spectroscopy. In summary, the rPTPetaCD is stable at 18 degrees C, properly folded, and fully active, which makes it a suitable candidate for structural and functional studies.