Catimel, BrunoTeh, TrazelFontes, Marcos R. M. [UNESP]Jennings, Ian G.Jans, David A.Howlett, Geoffrey J.Nice, Edouard C.Kobe, Bostjan2014-05-272014-05-272001-09-07Journal of Biological Chemistry, v. 276, n. 36, p. 34189-34198, 2001.0021-9258http://hdl.handle.net/11449/66582Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.34189-34198engisoproteinkaryopherinligandnuclear proteinnucleoplasminphosphoproteinkaryopherin alphavirus large T antigenactive transportanimalbiological modelcell nucleuschemical structurechemistrycircular dichroismdimerizationEscherichia coligenetic procedureskineticsmetabolismmousepeptide synthesisphysiologyprotein bindingprotein tertiary structuretimeultracentrifugationX ray crystallographyanimal cellbiosensorcell interactioncomplex formationconformational transitioncrystallographymolecular interactionnonhumannuclear importnucleocytoplasmic transportpriority journalprotein domainprotein localizationreceptor affinitystoichiometryActive Transport, Cell NucleusAnimalsBiosensing TechniquesCell NucleusCircular DichroismCrystallography, X-RayDimerizationKaryopherinsKineticsLigandsMiceModels, BiologicalModels, MolecularNuclear ProteinsPeptide BiosynthesisPhosphoproteinsProtein BindingProtein IsoformsProtein Structure, TertiaryTime FactorsUltracentrifugationSimiaeSimian virusSimian virus 40AnimaliaComplexationDimersElectrophoresisMonomersProteinsNuclear localization sequences (NLS)BiochemistryArtigo10.1074/jbc.M103531200Acesso restrito2-s2.0-0035823482