ABSTRACT
Methyltransferases play an important role in the post-transcriptional maturation of most ribonucleic acids. The modification of spliceosomal UsnRNAs includes N2-dimethylation of the m(7)G cap catalyzed by trimethylguanosine synthase 1 (TGS1). This 5'-cap hypermethylation occurs during the biogenesis of UsnRNPs as it initiates the m(3)G cap-dependent nuclear import of UsnRNPs. The conserved methyltransferase domain of human TGS1 has been purified, crystallized and the crystal structure of this domain with bound substrate m(7)GpppA was solved by means of multiple-wavelength anomalous dispersion. Crystal structure analysis revealed that m(7)GpppA binds via its adenosine moiety to the structurally conserved adenosylmethionine-binding pocket, while the m(7) guanosine remains unbound. This unexpected binding only occurs in the absence of AdoMet and suggests an incomplete binding pocket for the m(7)G cap which is caused by the N-terminal truncation of the protein. These structural data are consistent with the finding that the crystallized fragment of human TGS1 is catalytically inactive, while a fragment that is 17 amino acids longer exhibits activity.
Subject(s)
Methyltransferases/chemistry , RNA Cap Analogs/metabolism , RNA Caps/metabolism , Amino Acid Sequence , Binding Sites , Catalysis , Catalytic Domain , Conserved Sequence , Crystallography, X-Ray , Humans , Methylation , Methyltransferases/metabolism , Models, Molecular , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RNA Processing, Post-Transcriptional , S-Adenosylmethionine/metabolismABSTRACT
We synthesized biarylalanine-containing hydroxamic acids and tested them on immunoprecipitated HDAC1 and HDAC6 and show a subtype selectivity for HDAC6 that was confirmed in cells by Western blot (tubulin vs histones). We obtained an X-ray structure with a HDAC6-selective inhibitor with the bacterial deacetylase HDAH. Docking studies were carried out using HDAC1 and HDAC6 protein models. Antiproliferative activity was shown on cancer cells for selected compounds.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Phenylalanine , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Computer Simulation , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Histone Deacetylase 1 , Histone Deacetylase 6 , Histone Deacetylases , Histones , Humans , Hydroxamic Acids/chemical synthesis , Protein Binding , TubulinABSTRACT
The nuclear import of assembled spliceosomal subunits, the uridine-rich small nuclear ribonucleoprotein particles (U snRNPs), is mediated by a nuclear import receptor adaptor couple of importin beta (Imp beta) and snurportin1 (SPN1). In contrast to any other characterized active nuclear import, the Imp beta/SPN1/U snRNP complex does not require RanGTP for the terminal release from the nuclear basket of the nuclear pore complex (NPC). The crystal structure of Imp beta (127-876) in complex with the Imp beta-binding (IBB) domain of SPN1 (1-65) at 2.8-A resolution reveals that Imp beta adopts an open conformation, which is unique for a functional Imp beta/cargo complex, and rather surprisingly, it resembles the conformation of the Imp beta/RanGTP complex. As binding of RanGTP to Imp beta usually triggers the release of import complexes from the NPC, we propose that by already mimicking a conformation similar to Imp beta/RanGTP the independent dissociation of Imp beta/SPN1 from the nuclear basket is energetically aided.
Subject(s)
Active Transport, Cell Nucleus , RNA Cap-Binding Proteins/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Ribonucleoprotein, U1 Small Nuclear/metabolism , Spliceosomes/metabolism , beta Karyopherins/chemistry , Cell Nucleus , Crystallography, X-Ray , Humans , Nuclear Pore/metabolism , Nucleocytoplasmic Transport Proteins/chemistry , Nucleocytoplasmic Transport Proteins/physiology , Protein Binding , RNA Cap-Binding Proteins/physiology , Receptors, Cytoplasmic and Nuclear/physiology , beta Karyopherins/physiology , ran GTP-Binding Protein/chemistryABSTRACT
The nuclear import of H1 linker histones is mediated by a heterodimer of transport receptors, known as importinbeta and importin7. Interestingly, both importins separately interact with H1, but only as a dimer they facilitate the translocation through the nuclear pore. We identified the H1 binding site of importin7, comprising two extended acidic loops near the C terminus of importin7. The analysis of the H1 import complex assembly by means of isothermal titration calorimetry revealed that the formation of a receptor heterodimer in vitro is an enthalpy-driven process, whereas subsequent binding of H1 to the heterodimer is entropy-driven. Furthermore, we show that the importinbeta binding domain of importin7 plays a key role in the activation of importin7 by importinbeta. This process is allosterically regulated by importinbeta and accounts for a specific tuning of the activity of the importinbeta.importin7 heterodimer. The results presented here provide new insights into cellular strategies to even energy balances in nuclear import and point toward a general regulation of importinbeta-related nuclear import processes.
Subject(s)
Cell Nucleus/metabolism , Histones/metabolism , Karyopherins/metabolism , Models, Biological , Receptors, Cytoplasmic and Nuclear/metabolism , beta Karyopherins/metabolism , Active Transport, Cell Nucleus/physiology , Animals , Binding Sites , Chickens , Dimerization , Entropy , Humans , Protein Binding , Protein Structure, Secondary , Xenopus laevisABSTRACT
In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m3G)-cap. The import adaptor snurportin1 recognises the m3G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m3G-cap-binding domain of snurportin1 with bound m3GpppG at 2.4 A resolution, revealing a structural similarity to the mRNA-guanyly-transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.
Subject(s)
Nuclear Localization Signals/chemistry , RNA Cap-Binding Proteins/chemistry , RNA Caps/chemistry , Receptors, Cytoplasmic and Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/chemistry , Spliceosomes/chemistry , Active Transport, Cell Nucleus , Amino Acid Sequence , Animals , Binding Sites , Crystallography, X-Ray , Eukaryotic Initiation Factor-4E/chemistry , HeLa Cells , Humans , Methylation , Models, Molecular , Molecular Sequence Data , Nuclear Cap-Binding Protein Complex/chemistry , Nucleotidyltransferases/chemistry , Protein Conformation , RNA Cap-Binding Proteins/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Spectrometry, Fluorescence , Tryptophan/chemistry , Viral Proteins/chemistryABSTRACT
The nuclear import of spliceosomal UsnRNPs is mediated by the transport adaptor snurportin 1 (SPN1), which specifically recognizes the 2,2,7-trimethylguanosine (m(3)G) cap at the 5' end of UsnRNAs. Human SPN1 was overexpressed as a GST-fusion protein in Escherichia coli and purified to homogeneity. Since full-length SPN1 did not crystallize, limited proteolysis experiments were performed and stable digestion products were analyzed for functionality with respect to m(3)G cap-binding activity and subsequently used for crystallization trials. Well diffracting single crystals of a truncated SPN1 m(3)G cap-binding domain (residues 79-300) were obtained after two rounds of seeding. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 57.47, c = 130.09 A, alpha = beta = gamma = 90 degrees. Crystals contain one molecule in the asymmetric unit and diffract to a resolution limit of 2.9 A.
