Regulation of the classical nuclear import pathway through variation in the structure and function of importin α isoforms
The human genome encodes seven isoforms of the import adaptor importin &agr;, which binds to cargos bearing a classical Nuclear Localization Signal (NLSs). Despite remarkable in vivo specificity, little is known about the molecular basis by which NLS-cargos favor one isoform over the other. The influenza A virus polymerase subunit PB2 is a useful model cargo to study importin &agr; specificity because it is known to switch specificity from importin &agr;3 to importin &agr;7 in the adaptation from avian to mammalian strains. We used X-ray crystallography to obtain high resolution structures of PB2 in complex with three importin &agr; isoforms: mouse importin &agr;2 and the first solved structures of human importin &agr;3 and &agr;7. This structural work demonstrated for the first time that the NLS-binding surface is invariant among all isoforms, suggesting specificity must come from elsewhere. Using Molecular Dynamics simulations, we predicted that importin &agr;3 is much more flexible than other isoforms, while in vitro binding assays showed (i) that different isoforms have varying degrees of autoinhibition; and (ii) that the folded domain of PB2 contributed interactions in trans which contributed to high affinity binding to importin &agr;3. Overall, we conclude that these factors are the primary source of cargo specificity. In a separate study, we also examined the role of the nucleoporin Nup50 in the disassembly of PB2 from importin &agr;5. A previously reported structure of importin &agr;5 bound to PB2 showed a C-terminal extension of importin &agr;5, not seen in any other importin &agr; structures. We used X-ray crystallography to obtain a high resolution structure of importin &agr;5 bound to Nup50, which showed importin &agr;5 in the traditional, compact conformation seen in other importin &agr; structures. We used in vitro binding techniques to show that Nup50 and PB2 are able to bind importin &agr;5 simultaneously, suggesting that Nup50 on its own is not capable displacing cargo. These studies as a whole examine variation in the dynamic structures of importin &agr; isoforms to understand importin complex assembly and disassembly.
Pumroy, Ruth, "Regulation of the classical nuclear import pathway through variation in the structure and function of importin α isoforms" (2015). ETD Collection for Thomas Jefferson University. AAI3705104.