Biophysical characterization of interactions involving importin-alpha during nuclear import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin -alpha contains the NLS binding site, whereas importin-beta mediates the tr anslocation through the nuclear pore. We characterized the interactions inv olving importin-alpha during nuclear import using a combination of biophysi cal techniques (biosensor, crystallography, sedimentation equilibrium, elec trophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biose nsor). Association with importin-beta (stoichiometry, 1:1; K-D = 1.1 x 10(- 8) m) increases the affinity for NLSs; the importin-alpha/beta complex bind s representative monopartite NLS (simian virus 40 large T-antigen) and bipa rtite NLS (nucleoplasmin) with affinities (K-D = 3.5 x 10(-8) m and 4.8 x 1 0(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K-D = 1.7 x 10(-8) m; nuc leoplasmin NLS, K-D = 1.4 x 10(-8) m). The autoinhibitory domain (as a sepa rate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the i ntrasteric autoregulatory sequence of importin-alpha and provide a quantita tive description of the binding and regulatory steps during nuclear import.