Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import
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Data
2001-09-07
Autores
Catimel, Bruno
Teh, Trazel
Fontes, Marcos R. M. [UNESP]
Jennings, Ian G.
Jans, David A.
Howlett, Geoffrey J.
Nice, Edouard C.
Kobe, Bostjan
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Resumo
Proteins 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.
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Palavras-chave
isoprotein, karyopherin, ligand, nuclear protein, nucleoplasmin, phosphoprotein, karyopherin alpha, virus large T antigen, active transport, animal, biological model, cell nucleus, chemical structure, chemistry, circular dichroism, dimerization, Escherichia coli, genetic procedures, kinetics, metabolism, mouse, peptide synthesis, physiology, protein binding, protein tertiary structure, time, ultracentrifugation, X ray crystallography, animal cell, biosensor, cell interaction, complex formation, conformational transition, crystallography, molecular interaction, nonhuman, nuclear import, nucleocytoplasmic transport, priority journal, protein domain, protein localization, receptor affinity, stoichiometry, Active Transport, Cell Nucleus, Animals, Biosensing Techniques, Cell Nucleus, Circular Dichroism, Crystallography, X-Ray, Dimerization, Karyopherins, Kinetics, Ligands, Mice, Models, Biological, Models, Molecular, Nuclear Proteins, Peptide Biosynthesis, Phosphoproteins, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, Time Factors, Ultracentrifugation, Simiae, Simian virus, Simian virus 40, Animalia, Complexation, Dimers, Electrophoresis, Monomers, Proteins, Nuclear localization sequences (NLS), Biochemistry
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Journal of Biological Chemistry, v. 276, n. 36, p. 34189-34198, 2001.