Recognition of the TDP-43 Nuclear Localization Signal by Importin α1/β
Abstract
Cytoplasmic mislocalization of the TAR-DNA binding protein of 43 kDa (TDP-43) leads to large, insoluble aggregates that are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In this thesis work, we have characterized the interaction of the TDP-43 nuclear localization signal (NLS) with the classical nuclear import receptors importin α1 and importin β. We found that the NLS makes extensive contacts with importin α1, especially at the minor NLS-binding site. Correspondingly, the P2’ arginine (R83) at the minor site was necessary for binding to importin α1, while the P2 lysine (K97) at the major site was not. The R83 is near the globular N-terminal domain (NTD) of TDP-43, which we determined to dimerize at low concentration, and become increasingly oligomerized as NTD concentration increased. Complexation with importin α1 abolished this self-association of TDP-43, indicating that the importin α1 subunit possessed a novel disaggregating activity. Structural characterization of the TDP-43 interaction with importin α1 indicated that the C-terminus of importin α1 disrupts the TDP-43 NTD dimerization interface. We investigated the effect of phosphorylation within the TDP-43 NLS at T88, S91, and S92. Phosphomimetic mutation of these residues revealed that modification of T88 in the proximity of the minor binding site could reduce the affinity of the NLS for importin α1. Through molecular dynamics (MD) simulations, we found that phosphorylation of T88 destabilizes binding to importins by reducing the NLS backbone dynamics. Based on these data, we explain the pathogenic role of several post-translational modifications and mutations in the proximity of TDP-43 minor NLS site that are linked to disease and shed light on the chaperone activity of importin α1/β.
Subject Area
Biochemistry
Recommended Citation
Doll, Steven Gardner, "Recognition of the TDP-43 Nuclear Localization Signal by Importin α1/β" (2022). ProQuest ETD Collection - Thomas Jefferson University. AAI29254944.
https://jdc.jefferson.edu/dissertations/AAI29254944
