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[摘要]：Nuclear import of the pentameric histone chaperone nucleoplasmin (NP) is mediated by importin alpha, which recognizes its nuclear localization sequence (NLS), and importin beta, which interacts with alpha and is in charge of the translocation of the NP/alpha/beta complex through the nuclear pore. Herein, we characterize the assembly of a functional transport complex formed by full-length NP with importin alpha/beta. Isothermal titration calorimetry (ITC) was used to analyze the thermodynamics of the interactions of importin alpha with beta, alpha with NP, and the alpha/beta heterodimer with NP. Our data show that binding of both importin alpha and alpha/beta to NP is governed by a favorable enthalpic contribution and that NP can accommodate up to live importin molecules per NP pentamer. Phosphomimicking mutations of NP, which render the protein active in histone chaperoning, do not modulate the interaction with importin. Using small-angle X-ray scattering, we model the alpha/beta heterodimer, NP/alpha, and NP/alpha/beta solution structures, which reveal a glimpse of a complete nuclear import complex with an oligomeric cargo protein. The set of alternative models, equally well fitting the scattering data, yields asymmetric elongated particles that might represent consecutive geometries the complex can adopt when stepping through the nuclear pore. |
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