FUS-NLS/Transportin 1 Complex Structure Provides Insights into the Nuclear Targeting Mechanism of FUS and the Implications in ALS

Abstract

The C-terminal nuclear localization sequence of FUsed in Sarcoma (FUS-NLS) is critical for its nuclear import mediated by transportin (Trn1). Familial amyotrophic lateral sclerosis (ALS) related mutations are clustered in FUS-NLS. We report here the structural, biochemical and cell biological characterization of the FUS-NLS and its clinical implications. The crystal structure of the FUS-NLS/Trn1 complex shows extensive contacts between the two proteins and a unique α-helical structure in the FUS-NLS. The binding affinity between Trn1 and FUS-NLS (wide-type and 12 ALS-associated mutants) was determined. As compared to the wide-type FUS-NLS (KD = 1.7 nM), each ALS-associated mutation caused a decreased affinity and the range of this reduction varied widely from 1.4-fold over 700-fold. The affinity of the mutants correlated with the extent of impaired nuclear localization, and more importantly, with the duration of disease progression in ALS patients. This study provides a comprehensive understanding of the nuclear targeting mechanism of FUS and illustrates the significance of FUS-NLS in ALS.

Phosphorylation of C-terminal tyrosine 526 in FUS impairs its nuclear import

“…It has been shown that residue Y526 in FUS is one of the most important residues for a strong interaction with TNPO1 (Niu et al, 2012;Zhang and Chook, 2012). In order to reassess the role of the C-terminal Y526 residue in the nuclear import of FUS, we generated proteins that had been tagged at the N-terminus with green fluorescent protein (GFP), these included full-length wildtype FUS (FUS-WT), FUS with the deleted C-terminal Y256 (E,F) HeLa cells were transfected with His 6 -GFP-tagged C-terminal fragments (amino acids 490-526) of wild-type FUS and of mutants ΔY, Y526A, Y526F and Y526E, and co-immunoprecipitation was performed.…”

“…Residues P525 and Y526 of FUS dock into the hydrophobic pocket in TNPO1 through hydrophobic contacts. In addition, a hydrogen bond is formed between the hydroxyl group of Y526 in FUS and the carboxylic group of residue D384 in TNPO1 (Niu et al, 2012;Zhang and Chook, 2012). Based on this data, we modelled the interaction between FUS-NLS phosphorylated at Y526 and TNPO1 using the software programs Coot (Emsley et al, 2010) and Pymol (DeLano Scientific LLC, San Carlos, CA; http://www.pymol.org) (Fig.…”

“…This NLS mediates an interaction with a nuclear import factor transportin 1 (TNPO1), enabling the transport of FUS through the nuclear membrane (Guttinger et al, 2004;Lee et al, 2006;Dormann et al, 2010). The majority of ALS-associated FUS mutations fall within the NLS, which impairs interaction with TNPO1 and nuclear import (Bosco et al, 2010;Dormann et al, 2010;Gal et al, 2011;Niu et al, 2012;Vance et al, 2013). Consequently ALS cases with FUS mutations have FUS immunoreactive cytoplasmic inclusions in neuronal and glial cells (Kwiatkowski et al, 2009;Vance et al, 2009;Blair et al, 2010;Rademakers et al, 2010;Mackenzie et al, 2011).…”

Delineating the relationship between amyotrophic lateral sclerosis and frontotemporal dementia: Sequence and structure-based predictions

“…The C-terminal part of FUS encodes for a non-classic NLS that is necessary for nuclear import. The crystal structure of the FUS-NLS bound to its nuclear import receptor Karyopherinβ2 (Kapβ2; also known as Transportin) has been reported (PDB ID: 4FDD [45] and 4FQ3 [46]). …”

“…It is the most common cause of dementia after Alzheimer's disease (AD). The prevalence and incidence rate of FTD among the people belonging to [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64] year age group have been estimated to be 10-20 per 100,000 and 3.5-4.1 per 100,000 per year, respectively [1][2][3]. ALS is a devastating neurodegenerative condition affecting the motor neurons from the brain and spinal cord and is usually fatal due to respiratory paralysis which develops within 1-5 years of symptom onset [4].…”