Human importin alpha 3 and its N-terminal truncated form, without the importin-beta-binding domain, are oligomeric species with a low conformational stability in solution
Por:
Diaz-Garcia, C, Hornos, F, Giudici, A, Camara-Artigas, A, Luque-Ortega, J, Arbe, A, Rizzuti, B, Alfonso, C, Forwood, J, Iovanna, J, Gomez, J, Prieto, M, Coutinho, A and Neira, J
Publicada:
1 jul 2020
Resumen:
Background: Eukaryotic cells have a continuous transit of macromolecules between the cytoplasm and the nucleus. Several carrier proteins are involved in this transport. One of them is importin alpha, which must form a complex with importin beta to accomplish its function, by domain-swapping its 60-residue-long N terminus. There are several human isoforms of importin alpha; among them, importin alpha 3 has a particularly high flexibility.
Methods: We studied the conformational stability of intact importin alpha 3 (Imp alpha 3) and its truncated form, where the 64-residue-long, N-terminal importin-beta-binding domain (IBB) has been removed (Delta Imp alpha 3), in a wide pH range, with several spectroscopic, biophysical, biochemical methods and with molecular dynamics (MD).
Results: Both species acquired native-like structure between pH 7 and 10.0, where Imp alpha 3 was a dimer (with an apparent self-association constant of similar to 10 mu M) and Delta Imp alpha 3 had a higher tendency to self-associate than the intact species. The acquisition of secondary, tertiary and quaternary structure, and the burial of hydrophobic patches, occurred concomitantly. Both proteins unfolded irreversibly at physiological pH, by using either temperature or chemical denaturants, through several partially folded intermediates. The MD simulations support the presence of these intermediates.
Conclusions: The thermal stability of Imp alpha 3 at physiological pH was very low, but was higher than that of Delta Imp alpha 3. Both proteins were stable in a narrow pH range, and they unfolded at physiological pH populating several intermediate species.
General significance: The low conformational stability explains the flexibility of Imp alpha 3, which is needed to carry out its recognition of complex cargo sequences.
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